CN116648460A

CN116648460A - Screening methods for effective target-E3 ligase combinations

Info

Publication number: CN116648460A
Application number: CN202180043191.8A
Authority: CN
Inventors: M·M·莫里斯
Original assignee: UMC Utrecht Holding BV
Current assignee: UMC Utrecht Holding BV
Priority date: 2020-03-05
Filing date: 2021-06-18
Publication date: 2023-08-25
Also published as: WO2021176034A1; CN115803345A; JP2023517010A; CA3169792A1; EP4114854A1; AU2021232625A1; US20230142972A1

Abstract

The present invention relates to a method for identifying an effective combination of a transmembrane E3 ubiquitin ligase and a membrane bound protein, wherein the combination is effective if the transmembrane E3 ubiquitin ligase is capable of reducing the surface level of the membrane bound protein upon forced dimerization with the membrane bound protein, preferably by ubiquitination of the membrane bound protein. The method of the invention comprises the step of exposing the cell to a heterobifunctional molecule, wherein the heterobifunctional molecule comprises a first binding domain capable of specifically binding to an extracellular portion of a transmembrane E3 ubiquitin ligase and a second binding domain capable of specifically binding to an extracellular portion of a membrane binding protein. The method further comprises the step of determining a decrease in the surface level of the membrane-bound protein. The invention also relates to a heterobifunctional molecule targeting an effective combination of a transmembrane E3 ubiquitin ligase and a membrane-bound protein.

Description

Screening methods for effective target-E3 ligase combinations

Technical Field

The invention relates to the field of molecular cell biology, in particular to the field of molecular targeted therapy and cancer cell biology. The present invention relates to a method for screening for an effective combination of a target membrane-binding protein and an E3 ubiquitin ligase, and a method for generating heterobifunctional molecules that simultaneously target these discovered effective combinations. Thus, the present invention also relates to the use of a heterobifunctional molecule that can bind both to a transmembrane ubiquitin ligase and to a membrane-bound protein for mediating membrane-bound protein internalization.

Background

The activation of the cell by plasma membrane intercalating receptors communicates with its environment, which capture external chemical signals and initiate intracellular signaling cascades to drive the cellular response. The availability of cell surface receptors is a key determinant of signal specificity and sensitivity, and the misregulation of these events is often associated with the development and progression of diseases such as, but not limited to, cancer, autoimmune diseases, neurological disorders, and inflammatory disorders, as well as treatment tolerance.

For example, mutant activation or overexpression of receptors is a recognized major cancer-promoting mechanism in a variety of tissues (e.g., EGFR, ERBB2, PDGFR, tgfβ R, IGFR1, GHR, FZD, LRP). The dependence of cancer cells on aberrant receptor activation has prompted the development of various neutralizing antibodies and small molecule inhibitors. Successful neutralization of receptor activity, however, requires the formation of an effective conjugate to a sufficient plasma concentration to exhibit high efficacy without inducing toxicity, which can be difficult in the case of non-covalent interactors. Furthermore, compensatory receptor stabilization or upregulation is the primary pathway for tolerance.

The cytoplasmic region of the membrane-bound receptor is post-translationally modified with ubiquitin and is rapidly removed from the cell surface by induced endocytosis. The internalized receptor then undergoes lysosomal degradation. In healthy stem cells, high levels of Wnt signaling drive the expression of two cognate membrane-bound ubiquitin ligases, RNF43 and ZNRF3, which are known to mediate ubiquitination and removal of Wnt receptors, frizzled (FZD), from the cell surface (Koo et al, nature 2012,488 (7413):665-9). Thus, this negative feedback loop is used to modulate the sensitivity of stem cells to Wnt by controlling the effective amount of Frizzled (FZD) receptors on the cell surface. The activity of RNF43/ZNRF3 on FZD is neutralized in stem cell niches (stem cell niches) by secreted protein R-spondin (RSPO) that forms a complex with the LGR4/5 receptor and RNF43/ZNRF3 (Hao et al, nature 2012,485 (7397):195-200). Next, the trimeric RSPO-LGR4/5-RNF43/ZNRF3 complex is removed from the cell surface, resulting in stable FZD receptor expression and increased Wnt signaling levels. Wnt signaling is often misregulated in cancer. Such cancers show increased expression of Wnt target genes (including RNF43 and ZNRF 3).

The E3 ubiquitin ligase recruits the E2 ubiquitin-binding enzyme that has loaded ubiquitin onto a protein substrate and assisted or directly catalyzed the transfer of ubiquitin to the protein substrate.

It is well known that receptor ubiquitination mediated by transmembrane ubiquitin E3 ligase leads to endocytosis and subsequent breakdown of the ubiquitinated substrate. It is well known in the art that this decomposition preferably occurs in lysosomes. Lysosomal degradation requires attachment of a monoubiquitin, polyubiquitin, lys11, lys29, lys48 or Lys63 linked polyubiquitin chain to a membrane bound receptor. This is in contrast to the activity of cytoubiquitin ligases that use mainly the proteasome degradation pathway (i.e., coupling of polyubiquitin chains linked via Lys11, lys29 or Lys48 to cytoplasmic target proteins).

Thus, transmembrane E3 ubiquitin ligases can interact with different members of the E2 enzyme family to selectively target membrane bound substrates. The ubiquitinated substrate will be internalized and subsequently degraded, preferably by lysosomal degradation.

There remains a strong need in the art for methods of effectively targeting and inhibiting the activity of membrane-bound receptors, particularly those involved in the development or progression of disease. There is a particularly strong need in the art for methods that effectively target and inhibit membrane-bound receptor activity involved in the development or progression of, for example, cancer, autoimmune diseases, neurological disorders, rare diseases and inflammatory disorders, as well as in the treatment of tolerability.

Disclosure of Invention

The invention is summarized in the following embodiments:

embodiment 1. A method for identifying an effective combination of a transmembrane E3 ubiquitin ligase and a membrane bound protein, wherein the combination is effective when the transmembrane E3 ubiquitin ligase is simultaneously bound to a heterobifunctional molecule, the transmembrane E3 ubiquitin ligase being capable of reducing the surface level of the membrane bound protein, preferably by ubiquitination of the membrane bound protein, and wherein the method comprises the steps of:

a) Providing a cell, wherein the cell expresses a transmembrane E3 ubiquitin ligase and a membrane-bound protein on its cell surface;

b) Exposing the cell to a heterobifunctional molecule, wherein the heterobifunctional molecule comprises:

i) A first binding domain capable of specifically binding to an extracellular portion of a transmembrane E3 ubiquitin ligase; and

ii) a second binding domain capable of specifically binding to an extracellular portion of a membrane-bound protein; and

c) Determining the surface level of the membrane-associated protein of the cell,

wherein a decrease in the surface level of the membrane bound protein indicates that the combination is an effective combination, and wherein the decrease is preferably a decrease compared to the surface level of the membrane bound protein of the cell prior to step b).

Embodiment 2. The method of embodiment 1, wherein the membrane-bound protein is a transmembrane protein.

Embodiment 3. The method of embodiment 1 or 2, wherein the transmembrane E3 ubiquitin ligase is selected from the group consisting of RNF43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF130 and RNF128.

Embodiment 4. The method of any of the preceding embodiments, wherein at least one of:

-the transmembrane E3 ubiquitin ligase comprises a first extracellular non-native epitope tag and wherein the first binding domain of the heterobifunctional molecule binds to the first non-native epitope tag; and

-the membrane-bound protein comprises a second extracellular non-native epitope tag, and wherein the second binding domain of the heterobifunctional molecule binds to the second non-native epitope tag.

Embodiment 5. The method of embodiment 4, wherein the first non-native epitope tag and the second non-native epitope tag are different tags.

Embodiment 6. The method of embodiment 4 or 5, wherein the first non-native epitope tag is at least one of an alpha tag and an E6 tag, and/or wherein the second non-native epitope tag is at least one of an alpha tag and an E6 tag.

Embodiment 7. The method of any of embodiments 4 to 6, wherein at least one of the first non-natural epitope tag and the second non-natural epitope tag is located at least one of the following positions:

transmembrane E3 ubiquitin ligase and Membrane binding protein

i) An N-terminal;

ii) C-terminal; and/or

iii) Extracellular loop region.

Embodiment 8. The method according to any of the preceding embodiments, wherein the heterobifunctional molecule is a bispecific antibody, preferably a bispecific nanobody.

Embodiment 9. The method of embodiment 8, wherein the first binding domain of the heterobifunctional molecule is anti-Alpha VHH and the second binding domain is anti-E6 VHH, or wherein the first binding domain of the heterobifunctional molecule is anti-E6 VHH and the second binding domain is anti-Alpha VHH.

Embodiment 10. The method according to any of the preceding embodiments, wherein the membrane-bound protein comprises a third non-native epitope tag, and/or wherein the transmembrane ubiquitin E3 ligase comprises a fourth non-native epitope tag, preferably wherein the third and/or fourth epitope tag is at least one of a His tag, a FLAG tag and a myc tag.

Embodiment 11. The method according to any of the preceding embodiments, wherein the cell surface level of the membrane bound protein in step c) is determined by detecting a protein on the cell surface, preferably by immunofluorescence.

Embodiment 12. The method according to any of the preceding embodiments, wherein the combination is effective when the cell surface level of the membrane bound protein is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least about 95% compared to the cell surface level of the membrane bound protein prior to step b), preferably by at least about 60%, 70%, 80%, 90% or at least about 95% compared to the cell surface level of the membrane bound protein prior to step b).

Embodiment 13. The method according to any one of embodiments 4 to 11, wherein in step a) a first cell and a second cell are provided, wherein

-the first cell expresses a first transmembrane E3 ubiquitin ligase and a first membrane-binding protein on its cell surface; and is also provided with

The second cell expresses a second transmembrane E3 ubiquitin ligase and a first membrane bound protein on its cell surface,

wherein the first transmembrane E3 ubiquitin ligase and the second transmembrane E3 ubiquitin ligase are different ligases comprising the same first extracellular non-native epitope tag;

Wherein in step b) the first cell and the second cell are exposed to a heterobifunctional molecule, wherein the heterobifunctional molecule comprises:

i) A first binding domain capable of specifically binding to a first non-native epitope tag; and

ii) a second binding domain capable of specifically binding to an extracellular portion of a membrane-bound protein, preferably to a second non-native epitope tag; and is also provided with

Wherein the surface level of the membrane-bound protein of the first cell and the second cell is determined in step c), and wherein the combination is effective when the cell surface level of the membrane-bound protein in the first cell is reduced by at least about 10%, 20%, 30%, 40%, 50% or at least about 60% compared to the cell surface level of the membrane-bound protein in the second cell after step b).

Embodiment 14. The method of embodiment 13, wherein a third, fourth or more cells are provided that express a third, fourth or more transmembrane E3 ubiquitin ligase and a first membrane bound protein, respectively, on their cell surfaces, wherein the transmembrane E3 ubiquitin ligase is a different ligase comprising the same first extracellular non-native epitope tag,

And wherein the combination is effective when the cell surface level of the membrane bound protein in the first cell is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least about 95% compared to the cell surface level of the membrane bound protein in the second, third, fourth and further cells after step b),

and/or wherein the method is performed in a multiplexed manner.

Embodiment 15. The method according to any of the preceding embodiments, wherein the decrease in the surface level of the membrane bound protein is determined by a decrease in the total amount of membrane bound protein in the cell, preferably determined by microscopy, biochemical analysis and/or FACS.

Embodiment 16. The method according to any one of the preceding embodiments, wherein the cells provided in step a) overexpress (optionally permanently overexpress) at least one of a transmembrane E3 ubiquitin ligase and a membrane-bound protein.

Embodiment 17. The method according to any one of the preceding embodiments, wherein the cells provided in step a) express transmembrane E3 ubiquitin ligase and membrane-bound protein at endogenous levels.

Embodiment 18. The method according to embodiment 17, wherein in the cell provided in step a), the genomic sequence encoding the transmembrane E3 ubiquitin ligase has been modified to incorporate sequences encoding the first and optionally the fourth non-native epitope tag.

Embodiment 19. The method according to embodiment 17 or 18, wherein in the cell provided in step a), the genomic sequence encoding the membrane bound protein has been modified to incorporate sequences encoding the second and optionally the third non-native epitope tag.

Embodiment 20. The method according to any of the preceding embodiments, wherein the heterobifunctional molecule comprises a peptide linker located between the first binding domain and the second binding domain, and wherein preferably the peptide linker is (GGGGS) n, wherein n is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably wherein n is 3 or 5.

Embodiment 21. A heterobifunctional molecule comprising a first binding domain and a second binding domain, wherein,

i) The first binding domain is capable of specifically binding to a transmembrane E3 ubiquitin ligase; and is also provided with

ii) the second binding domain is capable of specifically binding to a membrane binding protein,

and wherein the transmembrane E3 ligase and the membrane-bound protein are an effective combination as determined in the method of any one of embodiments 1 to 20.

Embodiment 22. The heterobifunctional molecule of embodiment 21, wherein the molecule binds to the extracellular portion of the transmembrane E3 ubiquitin ligase and the extracellular portion of the membrane-bound protein.

Embodiment 23. The heterobifunctional molecule of embodiments 21 or 22, wherein the membrane-bound protein is a receptor, preferably a receptor involved in at least one of cancer, autoimmune disease, neurological disorder, and inflammatory disorder.

Embodiment 24. The heterobifunctional molecule of any one of embodiments 21 to 23, wherein the heterobifunctional molecule is a bispecific antibody, preferably a bispecific nanobody.

Embodiment 25. The heterobifunctional molecule of any one of embodiments 21 to 24, for use as a medicament.

Definition of the definition

Various terms relating to the methods, compositions, formulations, uses, and the like of the present invention are used in the specification and claims. Unless otherwise indicated, these terms should have their ordinary meaning in the art to which the invention pertains. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the testing practice of the present invention, the preferred materials and methods are described herein.

Methods of carrying out the conventional techniques used in the methods of the present invention will be apparent to those skilled in the art. Practice of routine techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well known to those skilled in the art and are discussed in the following literature references: sambrook et al, molecular cloning.a. Laboratory Manual,2nd Edition,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y, 1989; ausubel et al Current Protocols in Molecular Biology, john Wiley & Sons, new York,1987and periodic updates; and the series Methods in Enzymology, academic Press, san Diego.

"a", "an" and "the": these singular terms include plural designations unless the context clearly indicates otherwise. Thus, "a" or "an" generally refers to "at least one. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

"about" and "approximately": when these terms refer to measurable values, such as amounts, durations, etc., these terms are meant to encompass variations from the specified values of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, even more preferably ±0.1%, as these variations are suitable for performing the disclosed methods. Furthermore, amounts, ratios, and other numerical values are sometimes presented in a range format. It is to be understood that such range format is used for convenience and brevity and should be interpreted flexibly to include the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, ratios in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 to 200, but also include individual ratios, such as about 2, about 3, and about 4, as well as sub-ranges, such as about 10 to about 50, about 20 to about 100, and so forth.

"and/or": the term "and/or" refers to a situation in which one or more of the situations may occur alone or in combination with at least one of the situations until all of the situations occur together.

"comprise": the term should be interpreted as inclusive and open-ended, rather than exclusive. In particular, the terms and variations thereof are meant to encompass a particular feature, step, or component. These terms should not be interpreted to exclude the presence of other features, steps or components.

"exemplary": the term is intended to be "used as an example, instance, or illustration" and should not be interpreted as excluding other configurations disclosed herein.

The term'Heterobifunctional molecules"defined herein as a molecule comprising two different functional binding domains. In particular, the heterobifunctional molecules of the invention have a first functional binding domain for binding to a transmembrane E3 ubiquitin ligase and a separate second functional binding domain for binding to a second molecule. As the name "heterobifunctional" has indicated, the second functional binding domain binds to a second molecule, wherein the second molecule is not the same molecule that can be bound by the first functional binding domain, i.e. not the same transmembrane E3 ubiquitin ligase. Preferably, the second functional binding domain does not bind to the transmembrane E3 ubiquitin ligase.

The term'Proteins"OR"Polypeptides"refers to a molecule consisting of a chain of amino acids, but not to a particular mode of action, size, three-dimensional structure or source. Thus, a "fragment" or "portion" of a protein may still refer to a "protein". The protein as defined herein and used in any of the methods as described herein may be an isolated protein. "isolated protein" refers to an egg that is no longer present in its natural environment in vitro or in a recombinant bacterial or plant host cellWhite matter. Preferably, the protein comprises more than 50 amino acid residues.

The term'Protein molecules"in this context is understood as a molecule comprising short chains of amino acid monomers linked by peptide (amide) bonds. The short chain of amino acid monomers comprises 2 or more amino acid residues. Preferably, the amino acid chain has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acid residues. Preferably no more than 100 amino acid residues. Preferably, the protein has no more than 50 amino acid residues in the molecule. Preferably, the protein molecule has about 2 to 100, 3 to 50, 4 to 40 or 5 to 30 or 6 to 20 amino acid residues. Preferably, the protein molecule has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues. Optionally, the protein molecule comprises one or more other organic moieties, such as, but not limited to, a linker moiety that generates a circularized protein molecule.

“Aptamer"preferably a nucleic acid molecule having a specific nucleotide sequence. The aptamer may comprise any suitable number of nucleotides. The aptamer may comprise RNA or DNA, or comprise ribonucleotide residues and deoxyribonucleotide residues. The aptamer may be single-stranded, double-stranded, or comprise double-stranded or triple-stranded regions. In addition, the aptamer may comprise chemically modified residues, for example, to increase its stability.

The length of the aptamer is typically between about 10 and about 300 nucleotides. More typically, the length of the aptamer is between about 30 and about 100 nucleotides.

The aptamer of a given target (i.e., transmembrane E3 ubiquitin ligase or another transmembrane protein) comprises a nucleic acid that can be identified from a candidate nucleic acid mixture using a method comprising the steps of: (a) Contacting the candidate mixture with the target, wherein nucleic acids having increased affinity for the target as compared to other nucleic acids in the candidate mixture are separable from the remainder of the candidate mixture; (b) Separating the affinity-increasing nucleic acid from the remainder of the candidate mixture; and (c) amplifying the affinity-increasing nucleic acid to produce an enriched mixture of nucleic acids, thereby identifying an aptamer to the target molecule.

Affinity interactions are recognized as a degree of problem; however, in this case, the "specific binding affinity" of an aptamer to its target means that the aptamer will typically bind to its target to a much greater extent than to other non-target components in the mixture or sample.

The aptamer has a specific binding region that is capable of forming a complex with a target molecule in an environment where other substances in the same environment do not complex with the nucleic acid. The specificity of binding can be determined by the relative dissociation constant (K _d ) Defined (as compared to the dissociation constant of the aptamer for other materials or generally unrelated molecules in the environment). Generally, the aptamer is K relative to its ligand _d The specific aptamer is relative to K of irrelevant or concomitant materials in the environment _d At least about 10 times smaller. Even more preferably, K _d Will be at least about 50 times smaller, more preferably at least about 100 times smaller, and most preferably at least about 200 times smaller.

In certain embodiments, the dissociation constant (K) of the aptamer that binds to the transmembrane protein _d ) Less than or equal to 1mM, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM or less than or equal to 0.1nM. In certain embodiments, the anti-transmembrane protein antibodies bind to epitopes that are conserved among different species.

In certain embodiments, the dissociation constant (K) of the aptamer that binds to transmembrane E3 ubiquitin ligase _d ) Less than or equal to 1mM, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM or less than or equal to 0.1nM. In certain embodiments, the anti-transmembrane protein antibodies bind to epitopes that are conserved among different species.

The term'Antibodies to"in its broadest sense" specifically includes, for example, monoclonal antibodies (including agonists and antagonists), neutralizing antibodies, full length or intact monoclonal antibodies, polyclonal antibodies, multivalent antibodies, single chain antibodies, and functional fragments of antibodies, including Fab, fab ', F (ab') 2, and Fv fragments, diabodies, triabodies, single domain antibodies (sdabs), heavy chain antibodies, nanobodies, so long as they exhibit the desired biological and/or immunological activity.

The term "immunoglobulin" (Ig) is used interchangeably herein with antibody. Antibodies may be human and/or humanized.

The term "anti-transmembrane E3 ubiquitin ligase antibody" specifically includes, for example, single anti-transmembrane E3 ubiquitin ligase monoclonal antibodies (including agonists and antagonists, preferably agonists), neutralizing antibodies, full length or intact monoclonal antibodies, polyclonal antibodies, naked antibodies, multivalent antibodies, single chain anti-transmembrane E3 ubiquitin ligase antibodies and anti-transmembrane E3 ubiquitin ligase antibody fragments, including Fab, fab ', F (ab') 2 and Fv fragments, diabodies, triabodies, single domain antibodies (sdabs), heavy chain antibodies and nanobodies, so long as they exhibit the desired biological and/or immunological activity. Preferred antibodies may be nanobodies. Preferably, the anti-transmembrane E3 ubiquitin ligase antibody specifically binds to E3 ubiquitin ligase as defined below.

The term "anti-transmembrane protein antibody" specifically includes, for example, single anti-transmembrane protein monoclonal antibodies (including agonists and antagonists, preferably antagonists), neutralizing antibodies, full-length or intact monoclonal antibodies, polyclonal antibodies, naked antibodies, multivalent antibodies, single chain anti-transmembrane protein antibodies and anti-transmembrane protein antibody fragments, including Fab, fab ', F (ab') 2 and Fv fragments, diabodies, triabodies, single domain antibodies (sdabs), heavy chain antibodies and nanobodies, so long as they exhibit the desired biological and/or immunological activity. Preferred antibodies may be nanobodies. Preferably, the anti-transmembrane protein antibody specifically binds to a transmembrane protein as defined below.

The term "anti-transmembrane E3 ubiquitin ligase antibody" or "antibody that binds to transmembrane E3 ubiquitin ligase" refers to an antibody that is capable of binding to transmembrane E3 ubiquitin ligase with sufficient affinity such that the antibody can be used as the first binding domain of a heterobifunctional molecule as defined herein. Preferably, the anti-transmembrane E3 ubiquitin ligase antibody binds to an unrelated protein to a degree of less than about 10% of the degree of binding of the antibody to transmembrane E3 ubiquitin ligase, e.g. as measured by Radioimmunoassay (RIA) or ELISA. In certain embodiments, the dissociation constant (K) of an antibody that binds to transmembrane E3 ubiquitin ligase _d ) Less than or equal to 1mM, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM or less than or equal to 0.1nM. At a certain positionIn some embodiments, the anti-transmembrane E3 ubiquitin ligase antibody binds to an epitope that is conserved in different species.

The term "anti-transmembrane protein antibody" or "antibody that binds to a transmembrane protein" refers to an antibody that is capable of binding to a specific or selected transmembrane protein with sufficient affinity such that the antibody can be used as the second binding domain of a heterobifunctional molecule as defined herein. Preferably, the anti-transmembrane protein antibody binds to an unrelated protein to less than about 10% of the binding of the antibody to the transmembrane protein, as measured by Radioimmunoassay (RIA) or ELISA. In certain embodiments, the dissociation constant (K _d ) Less than or equal to 1mM, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM or less than or equal to 0.1nM. In certain embodiments, the anti-transmembrane protein antibody binds to an epitope that is conserved among different species.

An antibody that "binds" an antigen of interest (i.e., a transmembrane E3 ubiquitin ligase or another transmembrane protein of interest) is one that binds the antigen with sufficient affinity such that the antibody can function as the first binding domain or the second binding domain, respectively, of a heterobifunctional molecule as defined herein.

The antibody used as the first binding domain or the second binding domain in the heterobifunctional molecule may be a basic 4-chain antibody. Such basic 4-chain antibody units are preferably heterotetrameric glycoproteins consisting of two identical light (L) chains and two identical heavy (H) chains (IgM antibodies consist of 5 basic tetrameric units and one other polypeptide called J chain, thus comprising 10 antigen binding sites, whereas secreted IgA antibodies can polymerize to form multivalent combinations comprising 2 to 5 basic 4-chain units and J chains).

For IgG, the 4-chain unit is typically about 150000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H chain and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain at the N-terminus (V _H ) It follows that the alpha and gamma chains each have three constant domains (C _H ) Mu and epsilon isoforms have four C's, respectively _H A domain. Each L chain has a variable domain at the N-terminus (V _L ) The other end has a constant domain (C _L )。V _L And V is equal to _H Aligned, and C _L And heavy chain (C) _H 1) Is aligned with the first constant domain of (c). Certain amino acid residues are believed to form an interface between the light and heavy chain variable domains. V (V) _H And V _L Paired together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, e.g., basic and Clinical Immunology,8th edition,Daniel P.Stites,Abba I.Terr and Tristram G.Parslow (eds.), appleton&Lange,Norwalk,CT,1994,page 71and Chapter 6。

The L chain of any vertebrate species can be divided into two distinct types, kappa and lambda, based on the amino acid sequence of its constant domain. According to its heavy chain (C) _H ) Amino acid sequences of constant domains, immunoglobulins can be assigned to different types or isotypes. Immunoglobulins are of five types: igA, igD, igE, igG and IgM, the heavy chains are designated α, δ, ε, γ and μ, respectively. According to C _H The relatively small differences in sequence and function, the gamma and alpha classes are further divided into subclasses, e.g., humans express the following subclasses: igG1, igG2, igG3, igG4, igA1, and IgA2.

"variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "V _H ". The variable domain of the light chain may be referred to as "V _L ". These domains are typically the most variable parts of an antibody and comprise antigen binding sites.

The term "variable" refers to certain fragments of a variable domain that vary widely in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed over the 110 amino acids range of the variable domains. In contrast, the V region consists of a relatively constant extension called a 15 to 30 amino acid Framework Region (FR) separated by shorter regions of polar variability called "hypervariable regions" (HVRs), each 9 to 12 amino acids long. The variable domains of the natural heavy and light chains each consist of four FR, using mainly β -sheet structures, joined by three hypervariable regions to form loops (in some cases forming part of) that connect the β -sheet structures. The hypervariable regions in each chain are tightly linked together by the FR and, together with the hypervariable regions in the other chain, contribute to the formation of the antibody antigen binding site (see Kabat et al Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD (1991)). The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as antibodies involved in Antibody Dependent Cellular Cytotoxicity (ADCC).

"intact" antibody is intended to mean an antibody comprising an antigen binding site and C _L And at least heavy chain constant domain C _H 1、C _H 2 and C _H 3. The constant domain may be a natural sequence constant domain (e.g., a human natural sequence constant domain) or an amino acid sequence variant thereof.

An "antibody fragment" comprises a portion of an intact antibody, preferably at least the antigen binding and/or variable regions of an intact antibody. Examples of antibody fragments include Fab, fab ', F (ab') 2, and Fv fragments; a diabody; a tri-antibody; linear antibodies (see U.S. Pat. No.5,641,870, example 2; zapata et al, protein Eng.8 (10): 1057-1062[1995 ]); a single chain antibody molecule; and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment comprises the antigen binding site of an intact antibody and thus retains the ability to bind antigen.

The term'Nanobody"are well known in the art. Nanobodies are V comprising or consisting of heavy chain-only antibodies _H An antibody fragment consisting of an H domain. The terms "nanobody," "single domain antibody," and "single domain antibody fragment" are used interchangeably herein. The single domain antibody fragment has a single monomer variable antibody domain, preferably having a molecular weight of about 12 to 15kDa. Nanobodies, such as whole antibodies, are capable of selectively binding to specific antigens. The nanobody can be derived from dromedarion, camel, llama, alpaca or shark And (5) fish. Preferred nanobodies are derived from camelidae (camelidae), preferably derivable from llama.

Papain digestion of antibodies results in two identical antigen binding fragments, called "Fab" fragments, and one residual "Fc" fragment, reflecting their ability to crystallize readily. Fab fragments consist of the entire L chain and the variable region domain of the H chain (V _H ) And a first constant domain of a heavy chain (C _H 1) Composition is prepared. Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen binding site.

Pepsin treatment of antibodies will produce a single large F (ab') 2 fragment, which corresponds approximately to two disulfide-linked Fab fragments with bivalent antigen binding activity, and still be able to crosslink the antigen. Fab' fragments differ from Fab fragments in that at C _H The carboxy terminus of domain 1 has an additional small number of residues, including one or more cysteines from the antibody hinge region. Fab '-SH is the name of Fab' herein, wherein the cysteine residues of the constant domain have free thiol groups. F (ab ') 2 antibody fragments were initially produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of two H chains held together by disulfide bonds. The effector function of antibodies is determined by the sequence of the Fc region, which is also the part recognized by Fc receptors (FcR) on certain cell types.

"Fv" is the smallest antibody fragment that contains the complete antigen recognition and antigen binding site. The fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in close non-covalent association. In single chain Fv (scFv) species, one heavy chain variable region domain and one light chain variable domain may be covalently linked by a flexible peptide linker, such that the light and heavy chains may bind in a "dimer" structure similar to a double chain Fv species. Folding of these two domains creates six hypervariable loops (3 loops for each of the H and L chains) that provide amino acid residues for antigen binding and confer

Specificity of antigen binding to antibodies. However, even a single variable domain (or half of an Fv comprising only three antigen-specific CDRs) has the ability to recognize and bind antigen, but with less affinity than the entire binding site.

"Single chain Fv", also abbreviated "sFv" or "scFv", is a polypeptide comprising a V linked in a single polypeptide chain _H And V _L Antibody fragments of antibody domains. Preferably, the sFv polypeptide further comprises a polypeptide sequence located at V _H Domain and V _L Polypeptide linkers between domains that enable sFv to form the desired structure for antigen binding. For a summary of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, rosenburg and Moore eds., springer-Verlag, new York, pp.269-315 (1994).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possible presence of small amounts of naturally occurring mutations. Monoclonal antibodies have high specificity against a single antigenic site, whereas polyclonal antibody preparations contain different antibodies against different determinants (epitopes). Monoclonal antibodies are advantageous because they can be synthesized without contamination by other antibodies. The modifier "monoclonal" is not to be construed as requiring antibody production by any particular method. For example, monoclonal antibodies useful as the first binding domain or the second binding domain in the heterobifunctional molecules of the invention can be prepared by the hybridoma method first described by Kohler et al, nature,256:495 (1975), or can be prepared in bacterial, eukaryotic, or plant cells using recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described in the following documents: clackson et al, nature,352:624-628 (1991) and Marks et al, J.mol.biol.,222:581-597 (1991).

Monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as long as they exhibit the desired biological activity (see U.S. Pat. No.4,816,567; and Morrison et al, proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primate" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (e.g., old world monkey, ape, etc.) and human constant region sequences.

A "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody that comprises minimal sequences derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as a mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity and capacity. In some cases, some Framework Region (FR) residues of the human immunoglobulin are substituted with corresponding non-human residues. In addition, the humanized antibody may comprise residues not found in the recipient antibody or the donor antibody. These modifications were made to further improve antibody performance. In general, humanized antibodies will typically comprise two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FR is that of a human immunoglobulin sequence. The humanized antibody optionally further comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See Jones et al, nature321:522-525 (1986) for more details; riechmann et al, nature 332:323-329 (1988); and Presta, curr.Op.struct.biol.2:593-596 (1992). See also the following review articles and references cited therein: vaswani and Hamilton, ann. Allergy, asthma and immunol.,1:105-115 (1998); harris, biochem. Soc. Transactions,23:1035-1038 (1995); hurle and Gross, curr.Op.Biotech.,5:428-433 (1994).

The terms "hypervariable region", "HVR" as used herein refer to a region of an antibody variable domain that is hypervariable in sequence, and/or that forms a structurally defined loop responsible for antigen binding. Typically, an antibody comprises six hypervariable regions; three at VH (H1, H2, H3) and three at VL (L1, L2, L3). Many descriptions of hypervariable regions are in use and are included herein. Hypervariable regions typically comprise amino acid residues from "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in VL, residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in VH when numbered according to the Kabat numbering system; kabat et al, sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md. (1991)); and/or those residues from "hypervariable loops" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in VL, residues 26-32 (H1), 52-56 (H2), and 95-101 (H3) in VH when numbered according to the Chothia numbering system; chothia and Lesk, J. Mol. Biol.196:901-917 (1987)); and/or those residues from the "hypervariable loops"/CDRs (e.g., residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in VL, residues 27-38 (H1), 56-65 (H2) and 105-120 (H3) in VH when numbered according to the IMGT numbering system; lefranc, M.P.et al Nucl. Acids Res.27:209-212 (1999), ruiz, M.et al Nucl. Acids Res.28:219-221 (2000)). Alternatively, one or more of the positions 28, 36 (L1), 63, 74-75 (L2) and 123 (L3) in the VL and positions 28, 36 (H1), 63, 74-75 (H2) and 123 (H3) in the VH of the antibodies have symmetrical inserts when numbered according to Honneger, A. And Plinkthun, A.J. (mol. Biol.309:657-670 (2001)). The hypervariable regions/CDRs of the antibodies of the invention are preferably defined and numbered according to the IMGT numbering system.

"framework" or "FR" residues are those variable domain residues other than the hypervariable region residues defined herein.

A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

An "agonist antibody" as used herein is an antibody that mimics at least one functional activity of a polypeptide of interest.

"binding affinity" meansOften refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an inherent binding affinity that reflects a 1:1 interaction between binding pair members (e.g., antibodies and antigens). The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K _d ) And (3) representing. Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies typically bind to antigen slowly and are prone to dissociation, while high affinity antibodies typically bind to antigen faster and for longer periods of time. Numerous methods of measuring binding affinity are known in the art, any of which may be used for the purposes of the present invention. Specific illustrative embodiments are described below.

“K _d "or" K _d The value "by using BIAcore ^TM -2000 or BIAcore ^TM -3000 (BIAcore, inc., piscataway, NJ), measured at 25 ℃ in about 10 to 50 Response Units (RU) by surface plasmon resonance assay with immobilized antigen CM5 chip. Briefly, carboxymethylated dextran biosensor chips (CM 5, BIAcore inc.) were activated with N-ethyl-N' - (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the instructions of the supplier. The antigen was diluted to 5. Mu.g/ml (about 0.2. Mu.M) with 10mM sodium acetate (pH 4.8) and then injected at a flow rate of 5. Mu.l/min to give about 10 coupled protein Response Units (RU). After antigen injection, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of antibodies or Fab (0.78 nM to 500 nM) were injected with 0.05% tween 20 (PBST) at 25 ℃ into PBS at a flow rate of about 25 μl/min. Binding Rate (k) _on ) Dissociation rate (k) _off ) A simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) was used to calculate by fitting the binding and dissociation sensorgrams simultaneously. Equilibrium dissociation constant (K) _d ) Calculated as k _off /k _on Ratio of the two components. See, e.g., chen, y., et al, (1999) j.mol Biol 293:865-881. If passing through the surface plasmon Daughter resonance analysis, binding rate exceeding 10 ⁶ M ^-1 S ^-1 The binding rate can be determined by using fluorescence quenching techniques that measure an increase or decrease in the fluorescence emission intensity (excitation = 295nM; emission = 340nM,16nM bandpass) of 20nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25 ℃ with an increase in antigen concentration measured in a spectrometer (e.g., a spectrophotometer equipped with stopped flow (Aviv Instruments) or 8000-series SLM-amp co spectrophotometer with stirred red tube).

"on rate" or "rate of binding" or "k" according to the invention _on "BIAcore may also be used using the same surface plasmon resonance technique as described above ^TM -2000 or BIAcore ^TM -3000 (BIAcore, inc., piscataway, nj).

Preferably, the antibody used as the first binding domain or the second binding domain in the heterobifunctional molecule does not significantly cross-react with other proteins.

The terms "antigen binding protein" and "binding domain" of the heterobifunctional molecules of the invention are used interchangeably herein.

The term "epitope" is the portion of a molecule that binds to the first binding domain or the second binding domain, respectively, of a heterobifunctional molecule of the invention. The term includes any determinant capable of specifically binding to an antigen binding protein (e.g., specifically binding to a first domain or a second domain of a heterobifunctional molecule as defined below). Epitopes can be contiguous or noncontiguous (e.g., amino acid residues in a polypeptide that are not contiguous with each other in the polypeptide sequence but are bound by an antigen binding protein in the context of a molecule). The epitope is preferably located on a transmembrane E3 ubiquitin ligase as defined herein or another transmembrane protein of interest as defined herein.

Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, sulfonyl or sulfate groups, and may have specific three dimensional structural characteristics and/or specific charged characteristics. In general, antibodies specific for a particular target antigen will preferentially recognize epitopes on the target antigen in a complex mixture of proteins and/or macromolecules.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, a human IgG heavy chain Fc region is generally defined as extending from amino acid residue position Cys226 or from Pro230 to its carboxy-terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombining nucleic acids encoding the heavy chain of the antibody.

The term "Fc region-containing antibody" refers to an antibody comprising an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody, or by recombining the nucleic acid encoding the antibody. Thus, a heterobifunctional molecule comprising an antibody having an Fc region according to the invention may comprise an antibody having K447 or K447 removed.

"amino acid sequence": the term refers to a protein or the sequence of amino acid residues within a protein. In other words, any order of amino acids within a protein may be referred to as an amino acid sequence.

"nucleotide sequence": the term refers to a nucleic acid or the sequence of nucleotides within a nucleic acid. In other words, any order of nucleotides within a nucleic acid may be referred to as a nucleotide sequence.

The terms "homology", "sequence identity", and the like are used interchangeably herein. Sequence identity is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences as determined by aligning the sequences. In the art, "identity" also refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, as determined by the match between strings of such sequences. "similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and conservative amino acid substitutions thereof with the sequence of the second polypeptide.

The term "complementarity" is defined herein as the sequence identity of a nucleotide sequence to a perfectly complementary strand (e.g., the second strand or the reverse strand). For example, a sequence that is 100% complementary (or fully complementary) is herein understood to have 100% sequence identity to the complementary strand, e.g., a sequence that is 80% complementary is herein understood to have 80% sequence identity to the (fully) complementary strand.

"identity" and "similarity" can be easily calculated by well known methods. "sequence identity" and "sequence similarity" can be determined by aligning two peptide or two nucleotide sequences using global or local alignment algorithms, which algorithm is chosen depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., needleman Wunsch) that optimally aligns sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., smith Waterman). Sequences may be said to be "substantially identical" or "substantially similar" when they have at least a certain minimum percentage of sequence identity (defined below), for example, when optimally aligned by the programs GAP or BESTFIT using default parameters. GAP uses Needleman and Wunsch global alignment algorithms to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of GAPs. When the two sequences are of similar length, global alignment is suitably used to determine sequence identity. Typically, GAP generation penalty = 50 (nucleotides)/8 (proteins), GAP extension penalty = 3 (nucleotides)/2 (proteins) using GAP default parameters. For nucleotides, the default scoring matrix used was nwsgapdna, and for proteins, blosum62 (Henikoff & Henikoff,1992, PNAS 89, 915-919). Sequence alignment and percent sequence identity scoring may use computer programs such as GCG Wisconsin package (version 10.3) (available from Accelrys inc.,9685 Scranton Road,San Diego,CA 92121-3752 USA); or using open source software such as the program "needle" in EmbossWIN version 2.10.0 (using the global Needleman Wunsch algorithm) or "water" (using the local Smith Waterman algorithm), with the same parameters as GAP, or using default settings (for "needle" and "water", and for protein and DNA alignment, default GAP opening penalty of 10.0, default GAP extension penalty of 0.5, default scoring matrices Blosum62 (for protein) and DNAFull (for DNA)). When the sequences have substantially different overall lengths, local alignments are preferred, such as those using the Smith Waterman algorithm.

Alternatively, the percent similarity or identity may be determined by searching a public database using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid sequences and protein sequences of the invention may be further used as "query sequences" to perform searches on public databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTN and BLASTX programs of Altschul, et al (1990) J.mol.biol.215:403-10 (version 2.0). BLAST nucleotide searches can be performed using the NBLAST program (score=100, word length=12) to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed using the BLASTx program (score=50, word length=3) to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gap alignments for alignment purposes, gap BLAST can be used as described in Altschul et al, (1997) Nucleic Acids Res.25 (17): 3389-3402. When using BLAST and gap BLAST programs, default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See National Center for Biotechnology Information homepage, web address: http:// www.ncbi.nlm.nih.gov/.

As used herein, the term "preventing" refers to preventing or reducing the recurrence, onset, progression or progression of a disease (preferably a disease as defined below), or preventing or reducing the severity and/or duration of the disease or one or more symptoms thereof.

As used herein, the terms "treat," "treatment," "therapy" and "therapy" may refer to any regimen, method and/or agent useful for preventing, treating, managing or ameliorating a disease (preferably a disease as defined below) or one or more symptoms thereof.

As used herein, the term "treatment" refers to reducing or ameliorating the progression, severity and/or duration of a disease (preferably a disease as defined below), and/or reducing or ameliorating one or more symptoms of the disease.

As used herein, the term "effective amount" refers to an amount of a therapy (e.g., a prophylactic or therapeutic agent, preferably a heterobifunctional molecule as defined herein) sufficient to reduce the severity and/or duration of a disease, ameliorate one or more symptoms thereof, prevent disease progression or cause disease regression, or to prevent the development, recurrence, onset or progression of a disease or one or more symptoms thereof, or enhance or improve the prophylactic and/or therapeutic effect of another therapy (e.g., another therapeutic agent). Preferably, the disease is a disease as defined below.

Detailed Description

The present invention relates to the inventive concept of targeting membrane bound protein internalization and subsequent degradation using heterobifunctional molecules. The heterobifunctional molecules of the invention may bind both transmembrane ubiquitin ligase and membrane-bound proteins, e.g. carcinomatous receptors. Inducing ubiquitin ligases close to the desired target transmembrane protein (i.e. "forced dimerization") will lead to ubiquitination of the target protein, which is subsequently removed from the cell surface and subsequently degraded. For example, as a result, cancer cell growth is impaired. Fig. 1 provides a schematic diagram of an exemplary embodiment of the present invention.

The advantages of this method include at least the following:

i) The heterobifunctional molecules of the present invention allow for a strong increase in potency compared to conventional "drug occupancy-based" therapies, requiring only sub-stoichiometric amounts of the molecule compared to conventional target molecules.

ii) the specific binding required for both proteins (i.e. transmembrane E3 ubiquitin ligase and membrane binding protein) also reduces potential off-target toxicity. Preferably, ubiquitin ligases are used that localize to the plasma membrane and show increased expression in cancer cells.

iii) Since it takes time to synthesize a sufficient amount of the novel transmembrane protein, targeted protein degradation can prolong the pharmacodynamic effect.

iv) the heterobifunctional molecule binds to the extracellular protein moiety and thus does not need to cross the cell membrane.

v) it is well known that cancer cells express large amounts of various types of transmembrane E3 ubiquitin ligases, such as RNF43 and ZNRF3 in cancer cells with self-renewing properties. In this case, 4 alleles produce proteins with ubiquitination activity, reducing the possibility of mutant inactivation and tolerance.

The inventors have also found that not all membrane bound proteins can be targeted effectively by any transmembrane E3 ubiquitin ligase, i.e. bringing the membrane bound protein into close proximity to the transmembrane E3 ubiquitin ligase does not necessarily result in removal of the membrane bound protein from the cell surface. Thus, in order to develop effective heterobifunctional molecules, screening methods should be used to determine those combinations that, when in close proximity to the transmembrane E3 ubiquitin ligase, result in efficient internalization of the membrane bound protein.

The present inventors have discovered an effective method of screening for an effective combination of a transmembrane E3 ubiquitin ligase and a membrane bound protein, e.g., a combination in which induction of the proximity of the transmembrane ubiquitin E3 ligase to the membrane bound protein ("forced dimerization") results in removal of the membrane bound protein from the cell surface. Using this direct approach, an effective heterobifunctional molecule can be constructed targeting an effective combination of transmembrane E3 ubiquitin ligase and membrane-bound protein.

A particular advantage of the methods described herein is that a single heterobifunctional molecule can be used to screen different combinations of transmembrane E3 ubiquitin ligases and membrane bound proteins, for example by using the same first epitope for all transmembrane E3 ubiquitin ligases and the same second epitope for all membrane bound proteins. This provides an objective method for determining effective binding without regard to any variability, e.g., variable binding affinity, that may exist between different heterobifunctional molecules.

Thus, in one aspect, the invention relates to a heterobifunctional molecule comprising a first binding domain and a second binding domain. The first binding domain is capable of specifically binding to a transmembrane E3 ubiquitin ligase, and the second binding domain is capable of binding to a specific membrane binding protein. The combination of transmembrane E3 ubiquitin ligase and membrane-bound protein is preferably identified using the screening methods described herein.

Simultaneous binding to the transmembrane E3 ubiquitin ligase and the membrane bound protein brings the two molecules into proximity with each other. Thus, the transmembrane E3 ubiquitin ligase can subsequently ubiquitinate the membrane-bound protein.

Preferably, therefore, the simultaneous binding of the heterobifunctional molecule to the transmembrane E3 ubiquitin ligase and the membrane bound protein results in ubiquitination of the membrane bound protein.

Ubiquitination is known to result in degradation of ubiquitinated proteins. Thus, it is also preferred that the simultaneous binding of the heterobifunctional molecule to the transmembrane E3 ubiquitin ligase and the membrane bound protein results in degradation of the membrane bound protein.

Simultaneous binding to the transmembrane E3 ubiquitin ligase and the membrane bound protein brings the two molecules into proximity with each other. Thus, the membrane-bound protein may be internalized, preferably subsequently degraded.

Screening method

Prior to the preparation of heterobifunctional molecules that bind to a native epitope (heterobifunctional molecules as defined herein for use in the treatment of disease), an effective combination of transmembrane E3 ubiquitin ligase and membrane-bound protein can first be identified. Preferably, the combination is considered to be an effective combination when the transmembrane E3 ubiquitin ligase is capable of reducing the surface level of the membrane bound protein (preferably by ubiquitination of the membrane bound protein), preferably when the E3 ligase and the membrane bound protein are in proximity to each other (i.e. there is preferably forced dimerization of the transmembrane E3 ubiquitin ligase and the membrane bound protein). Thus, preferably, a combination is considered to be an effective combination when the transmembrane E3 ubiquitin ligase is capable of reducing the surface level of the membrane bound protein (preferably by ubiquitination of the membrane bound protein) upon forced dimerization of the transmembrane E3 ubiquitin ligase and the membrane bound protein. Preferably, the transmembrane E3 ubiquitin ligase and the membrane-bound protein are in close proximity by simultaneous binding to the heterobifunctional molecule as defined herein. Thus, preferably, a combination is considered to be an effective combination when the transmembrane E3 ubiquitin ligase and the membrane bound protein are simultaneously bound to a heterobifunctional molecule (preferably a heterobifunctional molecule as defined herein) and the transmembrane E3 ubiquitin ligase is able to reduce the surface level of the membrane bound protein.

The present inventors have developed a method for efficient screening of suitable combinations of transmembrane E3 ubiquitin ligase and membrane binding protein, e.g. combinations that can be targeted efficiently by the heterobifunctional molecules defined herein.

In one aspect, the invention thus relates to a method for identifying an effective combination of a transmembrane E3 ubiquitin ligase and a membrane bound protein, wherein the combination is an effective combination when the transmembrane E3 ubiquitin ligase is capable of lowering the surface level of the membrane bound protein. Preferably, the combination is an effective combination when the transmembrane E3 ubiquitin ligase can reduce the surface level of the membrane bound protein by ubiquitination of the membrane bound protein, preferably followed by internalization of the ubiquitinated membrane bound protein. The internalized ubiquitinated membrane-bound protein may then be degraded, preferably in lysosomes. Preferably, the method comprises the steps of:

wherein a decrease in the surface level of the membrane-bound protein indicates that the combination is an effective combination. Preferably, the decrease is a decrease compared to the surface level of the membrane bound protein of the cell prior to step b). Preferably, the decrease in protein level is a decrease in protein level of a membrane bound protein compared to a same or similar cell not exposed to the heterobifunctional molecule, e.g. a decrease in protein level of a membrane bound protein in a cell provided in step a) of the method of the invention.

The invention also relates to a method for reducing the surface level of a cell membrane-associated protein. The method preferably comprises the steps of:

a) Providing a cell, wherein the cell expresses a transmembrane E3 ubiquitin ligase and a membrane-bound protein on its cell surface; and

b) Exposing the cell to a heterobifunctional molecule as defined herein. The heterobifunctional molecule comprises:

ii) a second binding domain capable of specifically binding to an extracellular portion of a membrane-bound protein.

The method preferably further comprises step c) of determining the surface level of the membrane-bound protein of the cell. The reduction is preferably a reduction compared to the surface level of the membrane bound protein of the cell prior to step b). Preferably, the decrease in protein level is a decrease in protein level of a membrane bound protein compared to a same or similar cell not exposed to the heterobifunctional molecule, e.g. a decrease in protein level of a membrane bound protein in a cell provided in step a) of the method of the invention.

The method is preferably an ex vivo method, preferably an in vitro method.

Step a): providing cells

In step a) of the method of the invention, cells are provided. Any suitable cell for expressing the transmembrane E3 ubiquitin ligase and membrane-bound protein may be used in the methods of the invention. The cell preferably expresses transmembrane E3 ubiquitin ligase and membrane-bound protein on its cell surface. Preferably, the cell is an immortalized cell, preferably a cell line, preferably a cancer cell line. The cell may be a bacterial, yeast, plant or animal cell. Preferably, the cell is an animal cell. Preferred animal cells are vertebrate cells, preferably rodent or primate cells, preferably mouse or human cells. The cells may be cell cultures, cell lines, biopsies and organoids, or a part thereof. Preferably, the cells are part of or derived from patient-derived tissue (preferably cultured patient-derived tissue). The cells are part of or derived from a biopsy or organoid (preferably a tumor organoid). The biopsy may be a resected biopsy, an incision biopsy or a core needle penetration biopsy. The organoids are preferably cancerous organoids. The organoids are preferably patient-derived organoids, preferably tumor organoids.

The preferred cells are human cells, preferably at least one of cancer cells, immune cells and neural cells. Preferred cells are human cell lines, preferably human cancer cell lines, human immune cell lines and/or human neural cell lines. The cell line may be an immortalized cell line. Preferably, the cell is a HEK293T cell.

The provided cells may express a membrane-bound protein and/or a transmembrane E3 ubiquitin ligase at endogenous levels, or may be modified to induce or increase expression of the membrane-bound protein and/or the transmembrane E3 ubiquitin ligase. Additionally or alternatively, the cell may be modified to express a transmembrane E3 ubiquitin ligase comprising one or more non-native epitope tags as defined herein and/or a membrane-bound protein comprising one or more non-native epitope tags as defined herein. The transmembrane E3 ubiquitin ligase comprising the (non-native) epitope and the membrane-bound protein comprising the (non-native) epitope are preferably expressed in the same cell.

The provided cells, preferably cell lines, may express at least one of a wild-type or "native" transmembrane E3 ubiquitin ligase and a wild-type membrane-bound protein. At least one of the wild-type transmembrane E3 ubiquitin ligase and the wild-type membrane-bound protein may be overexpressed in the cell. The cell may transiently overexpress at least one of a wild-type transmembrane E3 ubiquitin ligase and a wild-type membrane-bound protein. Alternatively, at least one of the wild-type transmembrane E3 ubiquitin ligase and the wild-type membrane-bound protein may be permanently overexpressed in the cell.

Alternatively or additionally, provided cells, preferably cell lines, may express at least one of an engineered transmembrane E3 ubiquitin ligase and an engineered membrane bound protein. The engineered transmembrane E3 ubiquitin ligase comprises a first and optionally a fourth non-native epitope tag as defined herein. The engineered membrane-bound protein comprises a second and optionally a third non-native epitope tag as defined herein. The provided cells can transiently overexpress at least one of an engineered transmembrane E3 ubiquitin ligase and an engineered membrane-bound protein. Alternatively, the cell may permanently overexpress at least one of an engineered transmembrane E3 ubiquitin ligase and an engineered membrane-bound protein.

Expression (optionally permanent expression) of at least one of the engineered transmembrane E3 ubiquitin ligase and the engineered membrane-bound protein may be achieved using any conventional means known to those skilled in the art. For example, permanent expression may be achieved by integrating an expression cassette expressing at least one of the (engineered) transmembrane E3 ubiquitin ligase and the (engineered) membrane bound protein into the genome of the cell, for example.

Expression of the transmembrane E3 ubiquitin ligase (optionally comprising a non-native epitope) may be controlled by its native promoter or a non-native promoter such as, but not limited to, a constitutively active promoter. Expression of the membrane-bound protein (optionally comprising a non-native epitope) may be controlled by its native promoter or a non-native promoter, such as, but not limited to, a constitutively active promoter.

Sequences encoding at least one of a transmembrane E3 ubiquitin ligase (optionally comprising a non-native epitope) and a membrane-bound protein (optionally comprising a non-native epitope) can be introduced into a cell for transient or permanent expression. Optionally, the coding sequence is contained in an expression cassette introduced into the cell. The expression cassette preferably further comprises one or more elements controlling the expression of the transmembrane E3 ubiquitin ligase and/or controlling the expression of the membrane bound protein. Preferred expression elements are natural or non-natural promoters. The expression cassette may be part of an expression vector. Preferred expression vectors are naked DNA, DNA complexes or viral vectors. Preferred naked DNA is a linear or circular nucleic acid molecule, such as a plasmid. Plasmids refer to circular double-stranded DNA circles into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. The DNA complex may be a DNA molecule coupled to any carrier suitable for delivering DNA into a cell. Preferred carriers are selected from: lipid complexes (lipoplex), liposomes, polymeric vesicles (polymersomes), multimers (polyplexes), viral vectors, dendrimers, inorganic nanoparticles, virosomes and cell-penetrating peptides.

The provided cells can be modified to express at endogenous levels a transmembrane E3 ubiquitin ligase containing a non-native epitope tag and a membrane-bound protein containing a non-native epitope tag. As a non-limiting example, sequences encoding the first and optionally the fourth non-native epitope tag may be incorporated into the genomic sequence of the provided cell. Furthermore, in the same cell, sequences encoding the second and optionally the third non-native epitope tag may be incorporated into the genomic sequence of the provided cell.

Thus, the genomic sequence of a provided cell encoding a transmembrane E3 ubiquitin ligase can be modified to incorporate sequences encoding the first and optionally the fourth non-native epitope tag. The modified genomic sequence preferably encodes and expresses a transmembrane E3 ubiquitin ligase comprising the first and optionally fourth non-native epitope tag as defined herein. Preferably, in the same provided cell, the genomic sequence encoding the membrane-bound protein may be modified to incorporate sequences encoding the second and optionally the third non-native epitope tag. The modified genomic sequence preferably encodes and expresses a membrane-bound protein comprising a second and optionally a third non-native epitope tag as defined herein.

Methods for targeting genomic modifications to incorporate sequences encoding the first, second, and optionally third and fourth non-natural epitope tags are well known to those skilled in the art and include, but are not limited to, generating double strand breaks at genomic positions to incorporate sequences encoding the first and optionally fourth non-natural epitope tags, or site-directed endonucleases to incorporate sequences encoding the second and optionally third non-natural epitope tags. A preferred site-directed nuclease is the CRISPR-Cas system. Preferably, the double strand break generated is unique to a single location in the genome. Alternatively, double strand breaks may be generated at two or more genomic locations, wherein at least one of the double strand breaks is located in or near the sequence encoding the transmembrane E3 ubiquitin ligase or in or near the sequence encoding the membrane bound protein to merge sequences encoding the first or second and optionally the third and/or fourth tag, respectively.

One skilled in the art will directly understand that additional sequences may be incorporated into the genome, such as, but not limited to, a selection cassette to select for modified cells. Such a selection cassette is preferably incorporated into the intergenic or intronic region, preferably the intron of the transmembrane ubiquitin E3 ligase and/or the intron of the membrane bound protein.

Thus, the first and optionally the fourth non-native epitope tag may be introduced into the genome of the cell by the step of introducing into the cell: i) A site-directed nuclease that generates a double-strand break in or near the sequence encoding a transmembrane E3 ubiquitin ligase, and ii) an oligonucleotide or donor plasmid comprising sequences encoding the first and optionally the fourth tag. The double strand break is preferably located such that the mature transmembrane E3 ubiquitin ligase comprises a first and optionally a fourth tag. Preferably, the double strand break is located such that the first and optionally the fourth tag is located between the signal peptide and the mature transmembrane ubiquitin E3 ligase. Preferably, the double strand break is located such that the first and optionally the fourth tag are located extracellular. Preferably, the double strand break is located such that at least one of the first and optional fourth tags is located at or near the N-terminus of the mature transmembrane ubiquitin E3 ligase. Alternatively or additionally, at least one of the first and optional fourth tags may be located at or near the C-terminus of the mature transmembrane ubiquitin E3 ligase. Alternatively or additionally, at least one of the first and optional fourth tags may be located in an extracellular loop region of the mature transmembrane ubiquitin E3 ligase.

Cells expressing a transmembrane ubiquitin ligase comprising the first and optionally fourth non-native epitope tag can be used in the screening methods defined herein. In this embodiment, the heterobifunctional molecule may comprise a first binding domain for specifically binding to a first non-native epitope tag and a second binding domain capable of binding to a native epitope present in the wild-type transmembrane protein.

Preferably, in the same cell, the second and optionally the third non-native epitope tag may be introduced into the genome of the cell by the step of introducing into the cell: i) A site-directed nuclease that produces a double-strand break in or near the sequence encoding the membrane-bound protein, and ii) an oligonucleotide or donor plasmid comprising a sequence encoding the second and optionally the third tag. Preferably, the double strand break is located such that the second and optionally the third tag is located extracellular. The double strand break is preferably located such that the mature membrane bound protein comprises a second and optionally a third tag at the N-terminus. Alternatively or additionally, at least one of the second and optional third tags may be located at or near the C-terminus of the membrane bound protein. Alternatively or in addition, at least one of the second and optional third tags may be located in an extracellular loop region of a membrane bound protein.

The oligonucleotide or donor plasmid preferably comprises sequences that promote homology directed repair.

Alternatively or in addition, the first, second, and optionally third and fourth non-native epitope tags can be introduced using CRISPR-Cas lead editing techniques.

Alternatively, CRISPR techniques, such as those described above, can be used to generate suitable controls for the methods as defined herein, such as, but not limited to, the generation of transmembrane E3 ubiquitin ligases lacking a functional ligase domain.

Step b): exposing the cells to a heterobifunctional molecule

The step b) of exposing the cell to the heterobifunctional molecule is preferably performed under conditions allowing the heterobifunctional molecule to bind to both the transmembrane E3 ubiquitin ligase and the transmembrane protein. Such conditions are well known to those skilled in the art. As a non-limiting example, the heterobifunctional molecule may be added directly to the cell culture medium.

The concentration of the heterobifunctional molecule used in step b) of the method of the invention may vary, for example the concentration may depend on the heterobifunctional molecule and/or the epitope present in the transmembrane E3 ubiquitin ligase and/or the membrane-bound protein. The concentration of the heterobifunctional molecule can be determined experimentally using standard techniques. Preferably, the concentration of the heterobifunctional molecule exposed to the cell is about 0.1nM to 1000nM, about 0.5nM to 500nM, about 5nM to 100nM, about 20nM to 80nM, or about 40nM to 60nM. The concentration of heterobifunctional molecules is preferably about 50nM. The heterobifunctional molecules described herein are capable of binding both a transmembrane E3 ubiquitin ligase and a membrane-bound protein. The transmembrane E3 ubiquitin ligase is preferably a transmembrane E3 ubiquitin ligase as described herein.

The membrane-binding protein is preferably a transmembrane protein. The transmembrane protein may be at least one of type I, type II and type III transmembrane proteins. The transmembrane protein may be a so-called "multi-transmembrane" protein. The transmembrane protein may be a transmembrane protein as described herein.

The exposure time of the cells to the heterobifunctional molecule is preferably at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, or 24 hours, or preferably at least 1, 2, 3, 4, 5, 6, or 7 days.

At least one of the transmembrane E3 ubiquitin ligase and the membrane-bound protein may be a wild-type protein, e.g. a protein naturally encoded in the genome and optionally present (expressed) in the provided cell. The transmembrane E3 ubiquitin ligase and the membrane-bound protein are preferably expressed in the same cell. Optionally, at least one wild-type protein is overexpressed in the provided cells. Thus, alternatively, the wild-type transmembrane E3 ubiquitin ligase used in the methods of the invention has induced or increased expression in the provided cells. Alternatively, the wild-type membrane-bound protein used in the methods of the invention has induced or increased expression in the provided cells. The heterobifunctional molecules used in the methods of the invention preferably comprise a first binding domain capable of binding to a (native) epitope present in the wild-type transmembrane E3 ubiquitin ligase and/or comprise a second binding domain capable of binding to a (native) epitope naturally present in the wild-type membrane binding protein, preferably the wild-type transmembrane protein.

Or, at least one of the transmembrane E3 ubiquitin ligase and the membrane-bound protein is not a wild-type protein. Preferably, the transmembrane E3 ubiquitin ligase comprises a first non-native epitope tag. Preferably, the first non-native epitope tag is located in the extracellular portion of the ubiquitin ligase. Thus, preferably, the first non-native epitope tag is exposed on the cell surface of the provided cell. Preferably, the first non-native epitope tag is located at or near:

i) The N-terminus of transmembrane E3 ubiquitin ligase;

ii) the C-terminus of a transmembrane E3 ubiquitin ligase; and/or

iii) Extracellular loop region of transmembrane E3 ubiquitin ligase.

When the transmembrane E3 ubiquitin ligase comprises a first non-native epitope tag, the heterobifunctional molecule preferably comprises a first binding domain that selectively binds to the first non-native epitope tag.

Preferably, the membrane-bound protein comprises a second non-native epitope tag. Preferably, the second non-native epitope tag is located in an extracellular portion of the membrane-bound protein. Thus, preferably, the second non-native epitope tag is exposed on the cell surface of the provided cell.

Preferably, the second non-natural epitope tag is located at or near:

i) The N-terminus of the membrane-bound protein;

ii) the C-terminus of a membrane-bound protein; and/or

iii) Extracellular loop region of membrane-bound proteins.

When the membrane-bound protein comprises a second non-native epitope tag, the heterobifunctional molecule preferably comprises a second binding domain that selectively binds to the second non-native epitope tag.

In a preferred method of the invention, the cell is exposed to a heterobifunctional molecule, wherein the heterobifunctional molecule comprises:

i) A first binding domain capable of specifically binding to a first non-native epitope tag located in an extracellular portion of a transmembrane E3 ubiquitin ligase; and

ii) a second binding domain capable of specifically binding to a second non-native epitope tag located in an extracellular portion of the membrane bound protein.

"unnatural epitope tag" is herein understood to be an epitope that is not normally present in wild-type native proteins. The terms "epitope" and "epitope tag" are used interchangeably herein.

The first non-native epitope tag is preferably located in the extracellular portion of the transmembrane E3 ubiquitin ligase. Where the transmembrane E3 ubiquitin ligase comprises an extracellular N-terminal portion, the first epitope, preferably the first unnatural epitope, is preferably located at or near the original N-terminus of the transmembrane E3 ubiquitin ligase. Optionally, there are additional amino acid residues located between the original N-terminus and the first epitope. Alternatively, about 10 to 100, 5 to 50, or about 1 to 10 amino acid residues are located between the original N-terminus of the transmembrane E3 ubiquitin ligase and the first selectable non-native epitope. These amino acid residues may be native to the transmembrane E3 ubiquitin ligase or the transmembrane E3 ubiquitin ligase may be further extended by these additional amino acid residues.

Where the transmembrane E3 ubiquitin ligase comprises an extracellular C-terminal portion, the first epitope, preferably the first non-native epitope, is preferably located at or near the original C-terminus of the transmembrane E3 ubiquitin ligase. Optionally, there are additional amino acid residues located between the original C-terminal end and the first epitope. Alternatively, about 10 to 100, 5 to 50, or about 1 to 10 amino acid residues are located between the original C-terminus of the transmembrane E3 ubiquitin ligase and the first selectable non-native epitope. These amino acid residues may be native to the transmembrane E3 ubiquitin ligase or the transmembrane E3 ubiquitin ligase may be further extended by these additional amino acid residues.

Whether or not the transmembrane E3 ubiquitin ligase comprises an N-terminal and/or C-terminal extracellular portion, the first alternative non-native epitope tag may alternatively or additionally be located in the extracellular loop region of the transmembrane E3 ubiquitin ligase.

Alternatively or in addition, the first non-native epitope tag may extend one or more amino acid residues located near the intracellular portion of the transmembrane E3 ubiquitin ligase, such as but not limited to being located at or near the N-terminus and/or C-terminus of the original cell. In this embodiment, the non-native epitope tag may be extended by a transmembrane domain, wherein the transmembrane domain causes extracellular expression of the tag (preferably a peptide tag or a protein tag as defined herein). Non-limiting examples of such expansion tags are described in WO2012116076 and Brown et al (PLoS One,2013 Sep 2;8 (9): e73255 (the "Snorkel tag")), which is incorporated herein by reference. The transmembrane domain that extends the non-native epitope tag preferably has the sequence shown in FIG. 1 of Brown et al (supra). The Snorkel tag preferably has the sequence shown in fig. 1 of Brown et al (supra).

The second non-native epitope tag is preferably located in the extracellular portion of the membrane-bound protein. Where the membrane-bound protein comprises an extracellular N-terminal portion, the second epitope, preferably the second unnatural epitope, is preferably located at or near the original N-terminus of the membrane-bound protein. Optionally, there is an additional amino acid residue located between the original N-terminus and the second epitope. Alternatively, about 10 to 100, 5 to 50, or about 1 to 10 amino acid residues are located between the original N-terminus of the membrane bound protein and the second alternative non-native epitope. These amino acid residues may be native to the membrane-bound protein or the membrane-bound protein may be further extended by these additional amino acid residues.

Where the membrane-bound protein comprises an extracellular C-terminal portion, the second epitope, preferably the second unnatural epitope, is preferably located at or near the original C-terminus of the membrane-bound protein. Optionally, there is an additional amino acid residue located between the original C-terminal end and the second epitope. Alternatively, about 10 to 100, 5 to 50, or about 1 to 10 amino acid residues are located between the original C-terminus of the membrane bound protein and the second alternative non-native epitope. These amino acid residues may be native to the membrane-bound protein or the membrane-bound protein may be further extended by these additional amino acid residues.

Whether or not the membrane-bound protein comprises an N-terminal and/or C-terminal extracellular portion, the second alternative non-native epitope tag may alternatively or additionally be located in the extracellular loop region of the membrane-bound protein.

Alternatively or in addition, the second non-native epitope tag may extend one or more amino acid residues located near the intracellular portion of the membrane bound protein, such as, but not limited to, at or near the N-terminus and/or C-terminus of the original cell. In this embodiment, the non-native epitope tag may be extended by a transmembrane domain, wherein the transmembrane domain causes extracellular expression of the tag (preferably a peptide tag or a protein tag as defined herein). Non-limiting examples of such expansion tags are described in WO2012116076 and Brown et al (PLoS One,2013 Sep 2;8 (9): e73255 (the "Snorkel tag")), which is incorporated herein by reference. The transmembrane domain of the expansion tag preferably has the sequence shown in FIG. 1 of Brown et al (supra). The Snorkel tag preferably has the sequence shown in fig. 1 of Brown et al (supra).

Incorporation of the non-native epitope tag into the transmembrane E3 ubiquitin ligase and the membrane-bound protein, respectively, may be performed using any conventional molecular biology technique known in the art. The first and second epitope tags may be any suitable tag. The epitope tag may be a linear or conformational epitope. The tag is preferably a peptide tag or a protein tag. Preferably, the tag is a short amino acid sequence. The length of the first and/or second non-natural epitope tag is preferably between about 2 to 50, 3 to 40, 4 to 30, 5 to 20 or 8 to 15 amino acid residues. Preferably, the tag is an amino acid sequence that is directed against an antibody or antibody fragment (preferably nanobody) that can be produced using any conventional method known to those skilled in the art. The non-natural epitope tag may be a publicly available tag or a newly discovered sequence. The first tag and the second epitope tag may be the same or different tags. Preferably, the first and second epitope tags are different tags.

The first and/or second epitope tag may be an epitope tag selected from: alpha tag, E6 tag, V5 tag, VSV tag, avi tag, C tag, calmulin tag, polyglutamic acid tag, polyarginine tag, E tag, FLAG tag, HA tag, his tag, myc tag, NE tag, rho1D4 tag, S tag, SBP tag, softag 1, spot tag, strep tag, T7 tag, TC tag, ty tag and Xpress tag.

The first and/or second non-native epitope tag may be a protein tag selected from the group consisting of: GFP tag (green fluorescent protein), RFP tag (red fluorescent protein), YFP tag (yellow fluorescent protein), BFP tag (blue fluorescent protein), BCCP tag (biotin carboxyl carrier protein), glutathione-S-transferase tag, halo tag, SNAP tag, CLIP tag, HUH tag, maltose binding protein tag, nus tag, thioredoxin tag, fc tag, carbohydrate Recognition Domain (CRD) and CRDSAT tag.

The first and/or second non-native epitope tag may be extended by one or more amino acid residues that cause extracellular expression of the tag. The moiety that causes extracellular expression is preferably a transmembrane domain, preferably a transmembrane domain (TMD), preferably a TMD as shown in FIG. 1 of Brown et al (supra). The transmembrane domain may result in extracellular expression of a protein tag or peptide tag as defined herein.

The transmembrane domain may result in extracellular expression of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more protein tags or peptide tags as defined herein. Preferably, the transmembrane domain results in extracellular expression of at least one of a myc tag, FLAG tag, alpha tag and E6 tag. Preferably, the transmembrane domain results in extracellular expression of at least an E6 tag and a FLAG tag. Alternatively or in addition, the transmembrane domain may result in extracellular expression of at least an alpha tag and a myc tag.

The non-natural epitope tag may be selected from: alpha tag, E6 tag, myc tag, FLAG tag, his tag, V5 tag, VSV tag, GFP tag and RFP tag.

The first epitope tag may be an Alpha tag, preferably asAs described in et al (2019,Nature Communications,10 (1), 1-12). Alternatively, the first epitope tag may be a UBC6E tag (E6 tag), as described by Ling et al (2019,Molecular Immunology,114 (July), 513-523). The second epitope tag may be an Alpha tag, preferably such as +.>As described in et al, supra. Alternatively, the second epitope tag may be a UBC6E tag (E6 tag), as described by Ling et al (supra). Preferably, the first tag may be an Alpha tag and the second tag may be an E6 tag. Or alternatively, the first and second heat exchangers may be, The first tag may be an E6 tag and the second tag may be an Alpha tag.

Any suitable combination of epitope tag and corresponding antibody or antibody fragment recognizing said epitope may be used in the methods defined herein. Preferred antibody fragments are nanobodies. Thus, any suitable combination of an epitope and a corresponding nanobody recognizing said epitope may be used in the methods defined herein.

One skilled in the art is able to select the appropriate epitope-antibody or antibody fragment combination. For example, one skilled in the art can select suitable epitope-antibody or antibody fragment combinations known in the art. Alternatively or additionally, the epitope-antibody or antibody fragment combination may be a newly discovered combination and may be used in the methods defined herein.

The antibody or antibody fragment may be any suitable antibody or antibody fragment that specifically binds to the first or second epitope tag. Preferred antibody fragments are nanobodies. Preferably, the heterobifunctional molecule used in the method of the invention is a bispecific antibody, preferably a bispecific nanobody.

Preferably, in case the first epitope tag is an Alpha tag and the second epitope tag is an E6 tag, the first binding domain of the heterobifunctional molecule may comprise an anti-Alpha VHH and the second binding domain may comprise an anti-E6 VHH. Preferably, in the case where the first epitope tag is an E6 tag and the second epitope tag is an Alpha tag, the first binding domain of the heterobifunctional molecule may comprise an anti-E6 VHH (Ling et al, supra) and the second binding domain may comprise an anti-Alpha VHH # And the like, supra). Thus, a preferred epitope tag-binding domain combination is at least one of the following:

i) Alpha tag-anti-Alpha VHH (see e.g.And the like, supra); and

ii) E6 tag-anti-E6 VHH (see, e.g., ling et al, supra).

Preferred alpha tags have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 96. Preferred E6 tags have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 97. Preferred anti-alpha VHHs have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 98. Preferred CDR3 sequences of the anti-E6 VHH have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 99.

The membrane-bound protein may comprise a third non-native epitope tag. The third non-native epitope tag may be used to determine the protein level of a membrane bound protein, preferably at least one of the protein cell surface level, the total protein level, and the intracellular level of the membrane bound protein. The third non-native epitope tag is preferably located in the extracellular portion of the membrane-bound protein. Preferably, therefore, the third non-native epitope tag is exposed on the cell surface of the provided cell. Preferably, the third non-natural epitope tag is located at or near:

i) The N-terminus of the membrane-bound protein;

ii) the C-terminus of a membrane-bound protein; and/or

iii) Extracellular loop region of membrane-bound proteins.

The location of the third non-native epitope tag may be as described above for the second epitope tag. The third non-natural epitope tag may be located N-terminal and/or C-terminal to the second (non-natural) epitope tag.

The transmembrane E3 ubiquitin ligase may comprise a fourth non-native epitope tag. The fourth non-native epitope tag may be used to determine the protein level of transmembrane E3 ubiquitin ligase, preferably at least one of the protein cell surface level, total protein level and intracellular level of transmembrane E3 ubiquitin ligase. The fourth non-native epitope tag is preferably located in the extracellular portion of the transmembrane E3 ubiquitin ligase. Preferably, therefore, the fourth non-native epitope tag is exposed on the cell surface of the provided cell. Preferably, the fourth non-natural epitope tag is located at or near:

i) The N-terminus of transmembrane E3 ubiquitin ligase;

ii) the C-terminus of a transmembrane E3 ubiquitin ligase; and/or

iii) Extracellular loop region of transmembrane E3 ubiquitin ligase.

The fourth non-native epitope tag may be positioned as described above for the first epitope tag. The fourth non-natural epitope tag may be located N-terminal and/or C-terminal to the first (non-natural) epitope tag.

Thus, in embodiments, the transmembrane E3 ubiquitin ligase may comprise first and fourth non-native epitope tags and the membrane-bound protein may comprise second and third non-native epitope tags. The third and/or fourth non-natural epitope tag may be any conventional tag known to those skilled in the art for protein detection, such as, but not limited to, a peptide tag or a protein tag.

Alternatively, the peptide tag is selected from: alpha tag, E6 tag, V5 tag, VSV tag, avi tag, C tag, calmulin tag, polyglutamic acid tag, polyarginine tag, E tag, FLAG tag, HA tag, his tag, myc tag, NE tag, rho1D4 tag, S tag, SBP tag, softag 1, spot tag, strep tag, T7 tag, TC tag, ty tag and Xpress tag. Alternatively, the protein tag may be selected from: GFP tag (green fluorescent protein), RFP tag (red fluorescent protein), YFP tag (yellow fluorescent protein), BFP tag (blue fluorescent protein), BCCP tag (biotin carboxyl carrier protein), glutathione-S-transferase tag, halo tag, SNAP tag, CLIP tag, HUH tag, maltose binding protein tag, nus tag, thioredoxin tag, fc tag, carbohydrate Recognition Domain (CRD) and CRDSAT tag.

The third non-natural epitope tag may be selected from: myc tag, his tag, FLAG tag, V5 tag, VSV tag, HA tag, GFP and RFP. The fourth non-natural epitope tag may be selected from: myc tag, his tag, FLAG tag, V5 tag, VSV tag, HA tag, GFP and RFP.

Preferred combinations of the first tag and the fourth tag are myc tag and Alpha tag, myc tag and E6 tag, FLAG tag and Alpha tag, or FLAG tag and E6 tag. Preferred combinations of the second tag and the third tag are Flag tag and E6 tag, flag tag and Alpha tag, myc tag and E6 tag, or myc tag and Alpha tag.

The third and/or fourth non-native epitope tag may further comprise a moiety that causes extracellular expression of the tag. The moiety that causes extracellular expression is preferably a transmembrane domain, preferably a transmembrane domain (TMD), preferably a TMD as shown in FIG. 1 of Brown et al (supra). The extracellular expressed tag is preferably a peptide tag or a protein tag as defined herein.

Step c): determination of surface level of Membrane-bound proteins

The level or amount of membrane-bound protein may be determined using any conventional means known to those skilled in the art. Such means include, but are not limited to, microscopy, western immunoblotting, optionally quantitative immunofluorescence and/or quantitative western immunoblotting, cell surface biotinylation, FACS analysis, labeling of membrane bound proteins (e.g., SNAP labeling) with a cell impermeable fluorescent probe, and quantitative mass spectrometry.

The absolute amount or level of membrane-bound protein may be determined, for example, by directly comparing the levels of membrane-bound protein before or after exposure to the heterobifunctional molecule, for example, by determining the fluorescence intensity of the cell surface before and after exposure using, for example, fluorescence microscopy or FACS analysis.

For microscopic-based assays, fluorescent labeling of the membrane-bound proteins can be used to label surface-localized membrane-bound proteins, preferably in non-permeabilized cells. The total number of cells can be determined, for example, by staining the nuclei with DAPI or a similar dye. The average fluorescence intensity values of multiple images taken at the same magnification, laser power, optical settings and exposure time can be analyzed in ImageJ or similar analysis programs. Each image is preferably normalized to the number of stained nuclei, for example to its own DAPI value, the resulting value representing the relative amount of membrane-bound protein on each cell surface.

Alternatively or in addition, dual labelling of the membrane bound proteins may be used, first fluorescent labelling of the surface-localized membrane bound proteins in the non-permeant cells using a first epitope tag, followed by fluorescent labelling of the total membrane bound proteins in the permeant cells using a second epitope tag. For analysis by microscopy, the fluorescence intensity values of multiple images taken at the same magnification, laser power, optical settings and exposure time can be analyzed in ImageJ or similar analysis programs. For each image, the background level from untransfected cells is preferably subtracted, and for each condition, the average intensity value is preferred. The ratio of the average fluorescence intensity of the surface-localized proteins to the average fluorescence intensity of the total (membrane-bound) proteins is a measure of the relative amount of membrane-bound proteins at the surface (as described, for example, in Stuber et al, ACS Chem Biol,2019,14 (6): 1154-1163).

Alternatively or in addition, for analysis using flow cytometry, dead cells and non-cellular components are preferably excluded by forward and lateral scatter plots and based on negative DNA staining. Subsequently, the doublet cells are preferably excluded using a front scattering area relative to the height. The fluorescent signal may be quantified based on a threshold set, for example, based on the fluorescent intensity of untransfected cells as determined using FACSdiva, flowJo or similar analytical procedures. The ratio of the average fluorescence intensity of the surface localized proteins to the average fluorescence intensity of the total (membrane bound) proteins is a measure of the relative amount of membrane bound proteins at the surface (Stuber et al, supra).

Alternatively or in addition, the surface level of the membrane-bound protein may be determined based on the mature and immature forms of the protein. Membrane-bound proteins can undergo complex glycosylation and additional post-translational modifications during biosynthesis, resulting in having different Molecular Weights (MW), as visualized, for example, by western immunoblotting, where higher MW indicates that the mature form is present on the cell surface. The average pixel intensity of the bands representing the mature and immature forms of the membrane bound protein can be determined by western blot analysis software, including for example ImageQuant (Zeiss), imageJ or similar procedures. The ratio of the strength of the mature form of the membrane-bound protein to the total amount of membrane-bound protein is a measure of the relative amount of membrane-bound protein at the surface. (as described, for example, in Koo et al 2021, nature,2012;488 (7413): 665-9).

Alternatively or in addition, the relative levels or amounts of membrane bound proteins may be determined, for example, by comparison with the levels of home protein or total cellular protein before and after exposure, for example, using western blot analysis as described above.

Alternatively or in addition, the surface level of the membrane-bound protein may be determined by surface biotinylation. For this purpose, the surface proteins are labeled with cell-impermeable biotin and then all surface proteins are isolated by streptavidin pulldown. Subsequently, the collection of total proteins and surface localization proteins is analyzed, for example, using western blotting. The ratio of the intensity of the membrane bound protein in the surface pool to the intensity of the membrane bound protein in the total protein pool, as determined by analytical software such as ImageQuant (Zeiss), imageJ or similar procedure, is a measure of the relative amount of membrane bound protein at the surface. (Dubey et al, elife,2020;9:e54469;Hao et al,Nature 2012;485 (7397): 195-200).

Alternatively or in addition, a decrease in protein level of the membrane-bound protein may be determined by comparing different combinations of the membrane-bound protein with the transmembrane ubiquitin E3 ligase. As a non-limiting example, the protein level measured after forced dimerization of a membrane bound protein and a first transmembrane ubiquitin E3 ligase can be compared to the protein level measured after forced dimerization of the same membrane bound protein and a second transmembrane ubiquitin E3 ligase. This approach can determine the most effective combination of a membrane bound protein with a transmembrane ubiquitin E3 ligase, e.g.a transmembrane ubiquitin E3 ligase that results in the greatest reduction in surface levels of the membrane bound protein upon forced dimerization.

The transmembrane ubiquitin E3 ligase that causes a maximum decrease in surface level of the membrane bound protein upon forced dimerization preferably decreases the surface level by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to the transmembrane ubiquitin E3 ligase that causes a minimum decrease in surface level of the membrane bound protein upon forced dimerization or no decrease.

The method of the present invention may be performed directly in a multiplexed manner. As a non-limiting example, a first cell may be contacted with a first heterobifunctional molecule and a second cell may be contacted with a second heterobifunctional molecule. The first cell and the second cell are preferably physically separated, for example by holding them in separate wells. The first cell and the second cell may have the same genetic background. The first cell and the second cell may be the same cell.

The first cell may be exposed to a first heterobifunctional molecule and the second cell may be exposed to a second heterobifunctional molecule. The first binding domain and/or the second binding domain of the first heterobifunctional molecule and the second heterobifunctional molecule may be different.

The first binding domain and the second binding domain of the first heterobifunctional molecule and the second heterobifunctional molecule may be different.

The first binding domain of the first heterobifunctional molecule and the second heterobifunctional molecule may be different, while the second binding domain is not. The first binding domain of the first heterobifunctional molecule may selectively bind to a native or non-native epitope tag located in the first transmembrane E3 ubiquitin ligase. The first binding domain of the second heterobifunctional molecule may selectively bind to a native or non-native epitope tag located in the second transmembrane E3 ubiquitin ligase. The second binding domains of the first and second heterobifunctional molecules can selectively bind to native or non-native epitope tags located in the membrane-bound protein. This allows for a direct determination of the transmembrane ubiquitin E3 ligase which upon forced dimerization causes a maximum amplitude (i.e.most efficient) reduction of the surface level of the membrane bound protein.

The second binding domain of the first heterobifunctional molecule and the second heterobifunctional molecule may be different, while the first binding domain is not. The second binding domain of the first heterobifunctional molecule may selectively bind to a native or non-native epitope tag located in a membrane-bound protein. The second binding domain of the second heterobifunctional molecule may selectively bind to a native or non-native epitope tag located in the second membrane-bound protein. The first binding domain of the first and second heterobifunctional molecules preferably binds to a native or non-native epitope tag located in the transmembrane E3 ubiquitin ligase. This allows for the direct identification of membrane bound proteins that can be effectively reduced upon forced dimerization with certain transmembrane E3 ubiquitin ligases.

Alternatively, the first binding domain and the second binding domain of the first heterobifunctional molecule and the second heterobifunctional molecule are not distinct, but the first cell expresses a first transmembrane ubiquitin E3 ligase comprising a non-native epitope tag that can be bound by the first binding domain of the heterobifunctional molecule, and the second cell expresses a second transmembrane ubiquitin E3 ligase comprising the same non-native epitope tag. The first cell and the second cell preferably express a membrane-bound protein comprising a native or non-native epitope tag that is bound by the second binding domain of the heterobifunctional molecule. The first and second transmembrane ubiquitin E3 ligase are preferably two different transmembrane ubiquitin E3 ligases. The first transmembrane ubiquitin E3 ligase is preferably selected from the group consisting of: RNF43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF130, and RNF128. The second transmembrane ubiquitin E3 ligase is preferably selected from the group consisting of: RNF43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF130, and RNF128.

Alternatively, the first binding domain and the second binding domain of the first heterobifunctional molecule are indistinguishable, but the first cell expresses a first membrane-bound protein comprising a non-native epitope tag that can be bound by the second binding domain of the heterobifunctional molecule, and the second cell expresses a second membrane-bound protein comprising the same non-native epitope tag. The first cell and the second cell preferably express a transmembrane E3 ubiquitin ligase comprising a native or non-native epitope tag that can be bound by the first binding domain of the heterobifunctional molecule. The first and second membrane-bound proteins are preferably two different membrane-bound proteins.

Those of skill in the art will directly understand that the first cell and the second cell may be directly expanded to a third cell, a fourth cell, a fifth cell, etc. Likewise, the first and second heterobifunctional molecules may extend directly to the third heterobifunctional molecule, the fourth heterobifunctional molecule, the fifth heterobifunctional molecule, and so forth.

Preferably, the method of the invention comprises a step a) of providing a first cell and a second cell, wherein,

wherein the first and second transmembrane E3 ubiquitin ligases are different ligases comprising the same first extracellular non-native epitope tag;

ii) a second binding domain capable of specifically binding to an extracellular portion of a membrane binding protein, preferably to a second non-native epitope tag; and is also provided with

Wherein in step c) the surface level of the membrane bound protein of the first cell and the second cell is determined. The combination is preferably effective when the cell surface level of the membrane bound protein in the first cell is reduced by at least about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or about 100% compared to the cell surface level of the membrane bound protein in the second cell after step b). The effective combination is a combination of a first transmembrane E3 ubiquitin ligase and a membrane-bound protein.

When bound simultaneously to a heterobifunctional molecule, one combination of transmembrane E3 ubiquitin ligase and membrane-binding protein preferably results in a greater reduction in surface level of the membrane-binding protein than the other combination of transmembrane E3 ubiquitin ligase and (same) membrane-binding protein. This more effective combination is denoted herein as the combination in the "first cell". However, it will be readily appreciated by the person skilled in the art that the method involves differences between cells, for example that the combination is effective when the cell surface level of the membrane-bound protein in the second cell is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or about 100% compared to the cell surface level of the membrane-bound protein in the first cell after step b), as well as being viable. In this case, the second cell thus comprises the effective combination.

Preferably, a third, fourth or more cells are provided that express a third, fourth or more transmembrane E3 ubiquitin ligase and a first membrane bound protein, respectively, on their cell surfaces, wherein the transmembrane E3 ubiquitin ligase is a different ligase comprising the same first extracellular non-native epitope tag, and wherein the combination is effective when the cell surface level of the membrane bound protein in the first cell is reduced by at least about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or about 100% compared to the cell surface level of the membrane bound protein in the second, third, fourth and more cells after step b). Preferably, the method is performed in a multiplexed manner.

Using the method of the invention, the most effective combination can thus be determined. The most effective combination is preferably a combination of transmembrane E3 ubiquitin ligase and a membrane-bound protein, wherein the combination results in a maximum reduction of cell surface levels of the membrane-bound protein upon simultaneous binding to the heterobifunctional molecule. Preferably, the most effective combination is one that results in a reduction in cell surface level of the membrane-bound protein of at least about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least about 95% as compared to all other test combinations. When both the transmembrane E3 ubiquitin ligase and the membrane bound protein bind to the heterobifunctional molecule, the most effective combination of certain membrane bound proteins is preferably the transmembrane E3 ubiquitin ligase that most effectively mediates removal of the membrane bound protein from the cell surface compared to other transmembrane E3 ubiquitin ligases tested.

Furthermore, those skilled in the art will directly understand that the methods detailed herein are not limited by these embodiments, and that variants are likewise part of the present invention.

Protein levels of the membrane-bound protein may be determined before and after exposure to the heterobifunctional molecule as defined herein.

The protein level of the membrane bound protein may be determined after exposing the cells to the heterobifunctional molecule for at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, or 24 hours, or preferably at least 1, 2, 3, 4, 5, 6, or 7 days.

Preferably, the cell surface level of the membrane bound protein is reduced compared to the cell surface level of the membrane bound protein of the same cell not exposed to the heterobifunctional molecule (e.g. the cell provided in step a) of the method of the invention).

The terms "(protein) level" and "(protein) amount" are used interchangeably herein. Preferably, the cell surface level is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or by about 100% compared to the cell surface level of the membrane bound protein prior to step b) of the method of the invention. It should be understood here that a 100% decrease indicates that the transmembrane protein is no longer detectable on the cell surface. The reduction in cell surface protein levels can be determined by directly measuring the level or amount of cell surface residual protein after exposure to the heterobifunctional molecule. A preferred method for determining the level of membrane-bound proteins on the cell surface is immunofluorescence, preferably quantitative immunofluorescence.

The transmembrane ubiquitin E3 ligase is preferably a ubiquitin-like membrane binding protein. Thus, alternatively or in addition, a decrease in the surface level of the membrane-bound protein may be determined by determining the ubiquitination level of the membrane-bound protein. The increase in the level of ubiquitination is preferably inversely related to the cell surface level of the membrane-bound protein. Methods for determining the ability of the transmembrane E3 ubiquitin ligase to ubiquitinate the membrane bound protein include, but are not limited to, immunoprecipitation of the membrane bound protein, e.g., via a third epitope tag, and determination of binding to ubiquitin molecules using anti-ubiquitin antibodies. Alternatively or in addition, a tagged (preferably His-tagged) ubiquitin can be co-expressed with the membrane bound protein, and the level of ubiquitination can be determined using an anti-tagged (preferably anti-His-tagged) antibody. Additionally or alternatively, proteomic methods can be used to identify and quantify ubiquitin chains (reviewed in Fulzele and Bennett 2018 Methods Mol Biol).

Ubiquitination preferably results in internalization and degradation of the ubiquitinated membrane-bound protein. Thus, alternatively or in addition, the reduction of the surface level of the membrane bound protein may be determined by determining the total cellular protein level or amount of the membrane bound protein after exposure to the heterobifunctional molecule, e.g. after step b). The total protein level of the cell is preferably reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or by about 100% compared to the total protein level of the cell of the membrane bound protein prior to step b) of the method of the invention. It is understood herein that a 100% decrease indicates that the transmembrane protein is no longer detectable in the cell. Methods for determining total protein levels are well known and include, for example, standard biochemical analysis and FACS.

Alternatively or in addition, a decrease in the cell surface level of the membrane-bound protein may be determined by measuring an increase in the intracellular localization of the membrane-bound protein, preferably an increase in the endosomal localization of the membrane-bound protein. Ubiquitination of membrane-bound proteins preferably results in internalization of the membrane-bound protein. The internalized protein is then degradable, preferably in lysosomes. To determine an increase in intracellular protein localization of the protein, the method may include a step of inhibiting lysosomal conversion prior to the step of determining an increase in intracellular localization of the membrane bound protein, for example, but not limited to, by treating the cell with bafilomycin A1. Preferably, the intracellular localization of the membrane bound protein is increased by at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold or more compared to the intracellular localization of the membrane bound protein prior to step b) of the method of the invention.

Alternatively or in addition, reporter assays and/or downstream signaling assays may be used to determine a decrease in cell surface levels of the membrane bound protein. Such reporter assays and/or downstream signaling readouts are well known in the art and can be readily used in the methods of the invention.

Alternatively or in addition, co-localization of the membrane-bound protein and one or more lysosomal markers can be determined using standard techniques. The level of co-localization is preferably inversely related to the cell surface level of the membrane-bound protein.

The method defined above may also be considered as a method for selecting a combination of transmembrane E3 ubiquitin ligase and membrane-bound protein. Preferably, the methods defined herein are methods for selecting an effective combination of transmembrane E3 ubiquitin ligase and membrane-bound protein. Preferably, the combination is an effective combination when the transmembrane E3 ubiquitin ligase is capable of ubiquitinating the membrane bound protein when they are in close proximity. Ubiquitination of membrane-bound proteins preferably results in internalization of the protein. Preferably, the transmembrane E3 ubiquitin ligase and the membrane-bound protein are in close proximity by simultaneous binding to the heterobifunctional molecule as defined herein.

The method of the invention as defined above may also be considered as a method for determining the efficiency or efficacy of a heterobifunctional molecule (preferably a heterobifunctional molecule as defined herein) to reduce the surface level of a cell membrane-associated protein. The method preferably comprises the steps as described above. Preferably, the method comprises the steps of:

b) Exposing the cell to a heterobifunctional molecule, preferably a heterobifunctional molecule as defined herein.

The method preferably further comprises step c) of determining the surface level of the membrane-bound protein of the cell. The reduction is preferably a reduction compared to the surface level of the membrane bound protein of the cell prior to step b).

The method defined herein may also be considered as at least one of the following:

-a method for selecting an effective combination of a transmembrane E3 ubiquitin ligase and a membrane-bound protein (preferably a transmembrane protein), and wherein the combination is selected when the protein level of the transmembrane protein is reduced after step c).

-a method for screening for an effective combination of a transmembrane E3 ubiquitin ligase and a membrane-bound protein (preferably a transmembrane protein);

-a process for preparing a heterobifunctional molecule as defined herein, wherein the heterobifunctional molecule binds selectively to a selected combination of a transmembrane E3 ubiquitin ligase and a membrane binding protein (preferably a transmembrane protein);

-a method for determining the ability of a transmembrane E3 ubiquitin ligase to ubiquitinate a membrane bound protein, wherein the transmembrane E3 ligase is able to ubiquitinate the membrane bound protein when the protein level of the transmembrane protein decreases after step c);

-a method for targeting a membrane-bound protein for degradation by a heterobifunctional molecule; and

-a method for determining ubiquitination of a membrane-bound protein, and wherein a decrease in the surface level of the membrane-bound protein is indicative of ubiquitination of the membrane-bound protein, wherein the decrease is preferably a decrease compared to the surface level of the membrane-bound protein of the cell prior to step b).

As described above, the methods defined herein can be used to prepare potent heterobifunctional molecules, e.g., heterobifunctional molecules that target an effective combination of transmembrane E3 ubiquitin ligase and membrane-bound protein. The present invention therefore also relates to a heterobifunctional molecule, preferably a heterobifunctional molecule as defined herein, wherein the transmembrane E3 ubiquitin ligase and the membrane-bound protein selectively bound by the heterobifunctional molecule are selected using the method described above (preferred selection method). Thus, the method preferably comprises the steps of:

a) Providing a cell expressing a transmembrane E3 ubiquitin ligase and a membrane-bound protein on its cell surface, and wherein,

-the transmembrane E3 ubiquitin ligase comprises a first non-native epitope tag in the extracellular portion; and is also provided with

-the membrane-bound protein comprises a second non-native epitope tag in the extracellular portion;

-a first binding domain capable of specifically binding to the first non-native epitope tag; and

-a second binding domain capable of binding to the second non-native epitope tag;

c) Determining the surface level of the membrane-bound protein of the cell; and

d) The transmembrane E3 ubiquitin ligase and the transmembrane protein are selected when the surface level of the membrane-bound protein is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 100%, and wherein the reduction is a reduction compared to the surface level of the membrane-bound protein of the cell prior to step b).

In one aspect, the invention relates to a transmembrane E3 ubiquitin ligase comprising a first and optionally a fourth non-native epitope tag as defined herein.

In another aspect, the invention relates to a membrane-bound protein comprising a second and optionally a third non-native epitope tag as defined herein.

In one aspect, the invention relates to a combination of:

-a transmembrane E3 ubiquitin ligase comprising a first and optionally a fourth non-native epitope tag as defined herein; and

-a membrane-bound protein comprising a second and optionally a third non-native epitope tag as defined herein.

In one aspect, the invention relates to a host cell expressing a transmembrane E3 ubiquitin ligase comprising a first and optionally a fourth non-native epitope tag as defined herein. The host cell preferably also expresses a membrane-bound protein comprising a second and optionally a third non-native epitope tag as defined herein. Preferred host cells of the invention are the cells provided in step a) of the method of the invention.

Transmembrane E3 ubiquitin ligase bound by first binding domain

The invention also relates to a heterobifunctional molecule for use in the method of the invention. Furthermore, the present invention relates to a heterobifunctional molecule targeting an effective combination of transmembrane E3 ubiquitin ligase and a membrane binding protein, preferably an effective combination identified by the method of the invention.

The heterobifunctional molecules of the invention comprise a first binding domain capable of specifically binding to the extracellular portion of a transmembrane E3 ubiquitin ligase and a second binding domain capable of specifically binding to the extracellular portion of a membrane binding protein.

The first binding domain of the heterobifunctional molecule is capable of specifically binding to a transmembrane E3 ubiquitin ligase. Preferably, the transmembrane E3 ubiquitin ligase comprises a native epitope that can be specifically bound by a heterobifunctional molecule as defined herein, preferably when the heterobifunctional molecule is used as a medicament. Alternatively, the transmembrane E3 ubiquitin ligase may be engineered to comprise a non-native epitope, preferably a non-native epitope as defined herein. The non-native epitope may be specifically bound by a heterobifunctional molecule, preferably when the heterobifunctional molecule is used in the method of the invention, preferably the selection method of the invention.

The transmembrane E3 ubiquitin ligase may mediate ubiquitination and endocytosis of the membrane bound protein, i.e. of a protein bound by the second binding domain of the heterobifunctional molecule as defined herein.

Ubiquitination and endocytosis of the substrate preferably results in removal of the substrate from the cell surface. The internalized substrate may then be degraded. Thus, preferably, the transmembrane E3 ubiquitin ligase may mediate ubiquitination of membrane-bound proteins, removal and degradation from the cell surface, i.e. may mediate ubiquitination of proteins bound by the second binding domain of the heterobifunctional molecule as defined herein.

Preferably, therefore, the simultaneous binding of the heterobifunctional molecule to the transmembrane E3 ubiquitin ligase and the membrane-bound protein results in internalization of the membrane-bound protein, thereby removing the membrane-bound protein from the cell surface.

Preferably, the simultaneous binding of the heterobifunctional molecule to the transmembrane E3 ubiquitin ligase and the membrane bound protein results in internalization and degradation of the membrane bound protein. Thus, preferably, the transmembrane E3 ubiquitin ligase and the membrane bound protein are expressed in the same cell. Alternatively, at least one of the transmembrane E3 ubiquitin ligase and the membrane bound protein may be overexpressed in the cell.

Ubiquitination and degradation may be assessed using any suitable method known in the art. As a non-limiting example, ubiquitination and degradation can be assessed as described by Koo et al, nature (2012) (supra), which is incorporated herein by reference.

The substrate protein is selected to modify lysine residues by ubiquitin by interacting with the E3 ligase protein recruiting ubiquitin-bearing E2 enzyme (Clague MJ and Urb eS (2010), cell;43 (5): 682-5). This may result in the transfer (monoubiquitination) of a single ubiquitin molecule to a substrate, or in the coupling of another ubiquitin molecule to a previous ubiquitin molecule, e.g. through a lysine residue present in the previous ubiquitin molecule, forming a chain. Seven lysines of ubiquitin form different isopeptidic linkages that adopt different three-dimensional structures, and all of these are shown in eukaryotic cells (Xu et al (2009), cell 137, 133-145). The specific combination of E2 and E3 enzymes binding to the substrate determines the type of chain ligation.

Notably, lysosomal degradation may require a different pattern of ubiquitination of the substrate protein than proteasome degradation. For example, substrates labeled with a polyubiquitin chain linked with lysine 48 (Lys 48) often lead to proteasome targeting. Alternatively, substrates labeled with monoubiquitin, polyubiquitin, lys11, lys29, lys48 linked or Lys63 linked polyubiquitin are directed to lysosomes.

The degradation mediated by the transmembrane E3 ubiquitin ligase may be at least one of lysosomal degradation and proteasome degradation. Preferably, the degradation mediated by transmembrane E3 ubiquitin ligase is at least lysosomal degradation.

Thus, preferably, the simultaneous binding of the heterobifunctional molecule to the transmembrane E3 ubiquitin ligase and the membrane bound protein results in internalization of the membrane bound protein. Preferably, the simultaneous binding of the heterobifunctional molecule to the transmembrane E3 ubiquitin ligase and the membrane bound protein results in membrane bound protein internalization, and at least one of proteasome degradation and lysosomal degradation. Preferably, the simultaneous binding of the heterobifunctional molecule to the transmembrane E3 ubiquitin ligase and the membrane bound protein results in internalization of the membrane bound protein and lysosomal degradation.

Preferably, the transmembrane E3 ubiquitin ligase ubiquitinates the membrane-bound protein with a polyubiquitin chain linked with monoubiquitin, polyubiquitin, lys11, lys29, lys48 or Lys 63. Preferably, the transmembrane E3 ubiquitin ligase ubiquitinates the membrane-bound protein with a polyubiquitin chain linked with monoubiquitin, polyubiquitin or Lys 63. Preferably, the transmembrane E3 ubiquitin ligase ubiquitinates the membrane-bound protein with at least one of the polyubiquitin chains linked by Lys11, lys29, lys48 and Lys 63. Preferably, the transmembrane E3 ubiquitin ligase ubiquitinates the membrane-bound protein with a Lys63 linked polyubiquitin chain.

Many transmembrane E3 ubiquitin ligases exhibit tissue-specific expression or overexpression in one or more cancer types. Thus preferably, the transmembrane E3 ubiquitin ligase that can be bound by the heterobifunctional molecule defined herein is a transmembrane E3 ubiquitin ligase expressed in a selective tissue. As a non-limiting example, transmembrane E3 ubiquitin ligase RNF43 and ZNRF3 are selectively expressed in a population of adult stem cells in multiple tissues (e.g., without limitation, the intestine). As another non-limiting example, transmembrane E3 ubiquitin ligases MARCH1 and MARCH9 are expressed in immune cells.

Preferably, the transmembrane E3 ubiquitin ligase is expressed only in selective tissue (e.g., without limitation, cancer tissue).

Alternatively or in addition, a transmembrane E3 ubiquitin ligase that can be bound by a heterobifunctional molecule as defined herein is a transmembrane E3 ubiquitin ligase that exhibits expression (preferably overexpression) in one or more types of cancer.

Preferably, the transmembrane E3 ubiquitin ligase is preferably selected from: RNF43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF130, and RNF128. Preferably, the transmembrane E3 ubiquitin ligase is preferably selected from: RNF43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF145, RNFT1, RNF130, and RNF128. Preferably, the transmembrane E3 ubiquitin ligase is at least one of RNF43, RNF167, RNF128 and RNF 130. Preferably, the transmembrane E3 ubiquitin ligase is at least one of RNF43, RNF128 and RNF 167. Preferably, the transmembrane E3 ubiquitin ligase is at least one of RNF43 and RNF128.

The transmembrane E3 ubiquitin ligase may be overexpressed. As non-limiting examples, it is well known in the art that RNF43, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF167, RNF130 and RNF128 are expressed in increased in cancer.

In an embodiment, the heterobifunctional molecule comprises a first binding domain and a second binding domain, wherein,

i) The first binding domain is capable of specifically binding to a transmembrane E3 ubiquitin ligase, wherein the transmembrane E3 ubiquitin ligase is expressed, selectively expressed, or overexpressed in cancer tissue; and is also provided with

ii) the second binding domain is capable of specifically binding to a membrane-bound protein, wherein the membrane-bound protein is known or expected to be comprised in said cancer tissue,

wherein the simultaneous binding of the heterobifunctional molecule to the transmembrane E3 ubiquitin ligase and the membrane bound protein preferably results in ubiquitination and internalization of the transmembrane protein.

Preferably, the transmembrane E3 ubiquitin ligase and the membrane-bound protein are expressed in the same cell, preferably in the same cancer cell.

Preferably, both the E3 ubiquitin ligase and the membrane-bound protein are expressed in a cancer cell selected from the group consisting of: lung cancer, colorectal cancer, hepatocellular carcinoma, osteosarcoma, pancreatic cancer, gastric cancer, liver cancer, skin cancer, breast cancer, bladder cancer, ovarian cancer, esophageal cancer, thyroid cancer, cervical cancer, glioblastoma, squamous cell carcinoma, prostate cancer (gene expression profile), and intestinal cancer and/or metastases thereof.

Preferably, the membrane-bound protein is a transmembrane protein.

i) The first binding domain is capable of specifically binding to a transmembrane E3 ubiquitin ligase, wherein the transmembrane E3 ubiquitin ligase is expressed, preferably selectively expressed or overexpressed, in the immune cell; and is also provided with

ii) the second binding domain is capable of specifically binding to a membrane binding protein, wherein the membrane binding protein is expressed in the same immune cell, wherein the heterobifunctional molecule binds both to the transmembrane E3 ubiquitin ligase and to the membrane binding protein, preferably resulting in ubiquitination and internalization of the membrane binding protein.

Preferably, the membrane-bound protein is a transmembrane protein.

i) The first binding domain is capable of specifically binding to a transmembrane E3 ubiquitin ligase, wherein the transmembrane E3 ubiquitin ligase is expressed, preferably selectively expressed or overexpressed, in the neural cell; and is also provided with

ii) the second binding domain is capable of specifically binding to a membrane binding protein, wherein the membrane binding protein is expressed in the same neural cell, wherein the heterobifunctional molecule binds both to the transmembrane E3 ubiquitin ligase and to the membrane binding protein, preferably resulting in ubiquitination and internalization of the membrane binding protein.

Preferably, the membrane-bound protein is a transmembrane protein.

Preferably, the transmembrane E3 ubiquitin ligase has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29.

Preferably, the transmembrane E3 ubiquitin ligase is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30.

Preferably, the transmembrane E3 ubiquitin ligase is at least one of RNF43 and ZNRF 3. Proteins RNF43 and ZNRF3 preferably have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 1 and SEQ ID NO. 3, respectively. Preferably, the RNF43 and ZNRF3 proteins are encoded by sequences having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 2 and SEQ ID NO. 4, respectively.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF43. Preferably, the RNF43 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 1. Preferably, the RNF43 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 2.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is ZNRF3. Preferably, the ZNRF3 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 3. Preferably, the ZNRF3 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 4.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF13. Preferably, the RNF13 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 5. Preferably, the RNF13 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 6.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is AMFR. Preferably, the AMFR protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:7 (or SEQ ID NO: 51). Preferably, the AMFR protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 8 (or SEQ ID NO: 52).

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is MARCH1. Preferably, the MARCH1 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 9. Preferably, the MARCH1 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 10.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is MARCH4. Preferably, the MARCH4 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 11. Preferably, the MARCH4 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 12.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is MARCH2. Preferably, the MARCH2 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 13. Preferably, the MARCH2 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 14.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is MARCH8. Preferably, the MARCH8 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 15. Preferably, the MARCH8 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 16.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is MARCH9. Preferably, the MARCH9 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 17. Preferably, the MARCH9 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 18.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF149. Preferably, the RNF149 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 19. Preferably, the RNF149 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 20.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF145. Preferably, the RNF145 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 21. Preferably, the RNF145 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 22.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNFT1. Preferably, the RNFT1 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 23. Preferably, the RNFT1 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 24.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF167. Preferably, the RNF167 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 25. Preferably, the RNF167 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 26.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF130. Preferably, the RNF130 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 27. Preferably, the RNF130 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 28.

In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF128. Preferably, the RNF128 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 29. Preferably, the RNF128 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 30.

Proteins bound by the second binding domain

As detailed herein, the heterobifunctional molecules of the invention have a first binding domain capable of binding to a transmembrane E3 ubiquitin ligase (preferably a transmembrane E3 ubiquitin ligase as defined above).

The heterobifunctional molecules of the invention further comprise a second binding domain, wherein the second binding domain is capable of binding to a membrane-bound protein. Preferably, the membrane-bound protein comprises a native epitope that can be specifically bound by a heterobifunctional molecule as defined herein, preferably when the heterobifunctional molecule is used as a medicament. Alternatively, the membrane-bound protein may be engineered to comprise a non-native epitope, preferably a non-native epitope as defined herein. The non-native epitope may be specifically bound by a heterobifunctional molecule, preferably when the heterobifunctional molecule is used in the method of the invention, preferably the selection method of the invention.

Preferably, the protein that can be bound by the second binding domain of the heterobifunctional molecule is a protein that is at least partially exposed to the outside of the cell. The protein can be attached to a cell membrane from one side, or can span the whole membrane, namely the transmembrane protein. Preferably, the second binding domain is capable of specifically binding to a transmembrane protein.

As the term "heterobifunctional" has indicated, the membrane-bound protein that can be bound by the second binding domain is different from the transmembrane E3 ubiquitin ligase that can be bound by the first binding domain. Preferably, the second binding domain does not specifically bind and/or effectively bind to any transmembrane E3 ubiquitin ligase.

Preferably, the membrane-bound protein is a transmembrane protein, preferably at least one of a nutrient transporter, an ion channel and a cell surface receptor. Preferably, the second binding domain of the heterobifunctional molecule is capable of specifically binding to a transmembrane receptor. Preferably, the receptor is at least one of an ion channel coupled receptor, an enzyme coupled receptor, a G protein coupled receptor, and an Fc receptor. The second binding domain of the heterobifunctional molecule may bind to a monomeric form of the receptor and/or a dimeric form of the receptor. Additionally or alternatively, the second binding domain may bind to an inactive conformational receptor and/or an active conformational receptor.

The membrane-bound protein may be associated with, or involved in the development, progression or severity of a disease. The membrane-bound protein may be known or expected to be involved in cancer, autoimmune diseases, inflammatory disorders, neurological disorders, rare diseases, infectious diseases, and/or genetic diseases.

Preferably, the membrane-bound protein is not at least one of LGR4, LGR5 and LGR 6.

In a preferred embodiment, the membrane-bound protein is known or expected to be involved in neurological disorders. In preferred embodiments, the membrane-bound protein is known or expected to be involved in rare diseases.

In a preferred embodiment, the membrane-bound protein is known or expected to be involved in a disease selected from the group consisting of: fibular muscular atrophy (CMT), gaucher Disease (GD), anti-Mag peripheral neuropathy, CD 38-related neurodegenerative diseases, myostatin-related neuromuscular diseases, demyelinating diseases, MS, ALS, and gillin-Barre syndrome (GB). The CD 38-associated neurodegenerative disease may be at least one of ALS, MS, PD and AD. The myostatin related neuromuscular disease may be at least one of Duchenne type muscular dystrophy and cachexia.

In a preferred embodiment, the membrane-bound protein, preferably a membrane-bound receptor, is known or expected to be involved in cancer. "receptor involved in cancer" is herein understood to be a membrane-bound receptor that can directly or indirectly influence the malignancy of cancer.

In embodiments, a membrane-bound receptor involved in cancer may be a receptor that induces or enhances a malignant property of a cell upon activation or increased activity. For example, but not limited to, activation of the membrane-bound receptor may have an effect on at least one of stem cell characteristics, differentiation capacity, metabolism, viability, proliferation capacity, and immune evasion capacity. Activation of a receptor as used herein includes, but is not limited to, the receptor having one or more activating mutations, and/or the receptor being expressed and/or the availability of a receptor ligand being increased, and/or the receptor being less transformed, e.g., stabilized, on the cell membrane.

Additionally or alternatively, the membrane-bound receptor known or expected to be involved in cancer may be a receptor present on, for example, immune cells and/or stromal cells. As a non-limiting example, inhibiting a receptor present on an immune cell can result in immune cell activation to target tumor cells, and inhibiting a receptor present on a stromal cell can result in reduced tumor angiogenesis.

Thus, it is understood herein that the receptor involved in cancer may be a membrane-bound receptor present on tumor cells, and/or a membrane-bound receptor present on cells having a direct or indirect effect on tumor cells.

The phrase "a receptor associated with or involved in cancer" includes, but is not limited to, a proliferative disease (e.g., cancer or malignancy) or a precancerous condition (e.g., myelodysplastic syndrome, or pre-leukemia).

In one embodiment, the cancer associated with increased activation or activity of a membrane-bound receptor described herein is a hematological cancer. In one embodiment, the cancer associated with increased activation or activity of a membrane-bound receptor described herein is a solid cancer. Other diseases associated with increased activation or activity of the membrane-bound receptors described herein include, but are not limited to, for example, atypical cancers, malignant tumors, pre-cancerous conditions, or proliferative diseases associated with activation of the membrane-bound receptors described herein. Non-cancer related indications associated with increased activation or activity of membrane-bound receptors as described herein include, but are not limited to, for example, autoimmune diseases (e.g., lupus), inflammatory disorders (allergy and asthma), and transplantation.

Preferably, the membrane-bound receptor that can be bound by the second domain of the heterobifunctional molecule is a receptor involved in cancer, and thus preferably the activity of the receptor is increased, e.g. downstream signaling is increased. Downstream signaling is preferably increased in advantage over otherwise identical cells that do not have increased activation or activity of membrane-bound receptors. The increase in activity may be due to, but is not limited to, mutant activation of the receptor, upregulation of the receptor, increased stability of the receptor, and/or increased availability of the receptor ligand.

The receptor may be involved in a particular type of cancer. Alternatively, the receptor may be involved in a variety of different types of cancers. For example, the receptor may be involved in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more types of cancer. Alternatively or in addition, the receptor may be involved in cancer angiogenesis.

The receptor may be involved in solid or hematological cancers. The receptor may be involved in solid cancers. Preferably, the solid cancer is selected from the following: colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, hodgkin's disease, non-hodgkin's lymphoma, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, childhood solid tumor, bladder cancer, renal cancer or ureter cancer, renal pelvis cancer, central Nervous System (CNS) tumors, primary CNS lymphomas, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, combinations of said cancers, and metastatic lesions of said cancers.

The receptor may be involved in solid or hematological cancers. Preferably, the hematological cancer is selected from one or more of the following: chronic Lymphocytic Leukemia (CLL), acute leukemia, acute Lymphoblastic Leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic lymphoma (T-ALL), chronic Myelogenous Leukemia (CML), B-cell prolymphocytic leukemia, lymphoblastic plasmacytoid dendritic cell tumor, burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative disease, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-hodgkin's lymphoma, plasmablastoman lymphoma, plasmacytoid dendritic cell tumor, fahrenheit macroglobulinemia, or pre-leukemia.

Preferably, the membrane-bound protein that can be bound by the second binding domain of the heterobifunctional molecule is involved in a cancer selected from: colorectal cancer, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, prostate cancer, lung cancer, melanoma, leukemia, pancreatic cancer, and bladder cancer.

Preferably, the membrane-bound protein that is bound by the second binding domain of the heterobifunctional molecule is activated in colorectal cancer or increases activity in colorectal cancer. Preferably, the membrane-bound protein is at least one of EGFR, IGF1R, MET, LRP6, WLS and ERBB2.

Preferably, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is activated in hepatocellular carcinoma or increases activity in hepatocellular carcinoma. Preferably, the membrane-bound protein is LRP6.

Preferably, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is activated in colorectal cancer or increases activity in colorectal cancer. Preferably, the membrane-bound protein is LRP6 or WLS.

Preferably, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is activated in breast cancer, or increases activity in breast cancer. Preferably, the membrane-bound protein is WLS, EGFR or ERBB2.

Preferably, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is activated in esophageal cancer, or increases activity in esophageal cancer. Preferably, the membrane-bound protein is ERBB2 or VEGFR2.

Preferably, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is activated in gastric cancer, or increases activity in gastric cancer. Preferably, the membrane-bound protein is WLS, ERBB2 or VEGFR2.

Preferably, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is activated in leukemia, or increases activity in leukemia. Preferably, the membrane-bound protein is FLT3.

Preferably, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is activated in melanoma or increases activity in melanoma. Preferably, the membrane-bound protein is KIT.

Preferably, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is activated in, or increases activity in, non-small cell lung cancer. Preferably, the membrane-bound protein is EGFR or MET.

Preferably, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is activated in ovarian cancer, or increases activity in ovarian cancer. Preferably, the membrane-bound protein is EGFR.

Preferably, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is activated in pancreatic cancer, or increases activity in pancreatic cancer. Preferably, the membrane-bound protein is LRP6 or EGFR.

Preferably, the membrane-bound protein that can be bound by the second binding domain of the heterobifunctional molecule is selected from the group consisting of: tgfβr1, tgfβr2, EGFR, ERBB2, ERBB3, IGF1R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CMTM6, CMTM4, WLS, SLC7A5, and SLC16A7.

Preferably, the membrane-bound protein that is bindable by the second binding domain of the heterobifunctional molecule has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 84, 86, 88, 90, 92, 94, 100, 102 and 104.

Preferably, the membrane-bound protein bindable by the second binding domain of the heterobifunctional molecule is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 85, 87, 89, 91, 93, 95, 101, 103 and 105.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is tgfβr1 or tgfβr2.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is tgfβr1. Preferably, the TGF-beta R1 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 31. Preferably, the TGF-beta R1 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 32.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is tgfβr2. Preferably, the TGF-beta R2 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 33. Preferably, the TGF-beta R2 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 34.

Preferably, the second binding domain of the heterobifunctional molecule is capable of specifically binding to tgfβr2, whereas the first binding domain is capable of specifically binding to RNF 167.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is EGFR. Preferably, the EGFR protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 35. Preferably, the EGFR protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 36.

Preferably, the second binding domain of the heterobifunctional molecule is capable of specifically binding to EGFR, while the first binding domain is capable of specifically binding to RNF 167.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is ERBB2 or ERBB3.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is ERBB2. Preferably, the ERBB2 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 37. Preferably, the ERBB2 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 38.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is ERBB3. Preferably, the ERBB3 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 39. Preferably, the ERBB3 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 40.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is IGF1R. Preferably, IGF1R protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 41. Preferably, IGF1R protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 42.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is MET. Preferably, the MET protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 43. Preferably, the MET protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 44.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is VEGFR2. Preferably, the VEGFR2 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 45. Preferably, the VEGFR2 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 46.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is KIT. Preferably, the KIT protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 47. Preferably, the KIT protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 48.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is FLT3. Preferably, the FLT3 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 49. Preferably, the FLT3 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 50.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is PDGFRA. Preferably, the PDGFRA protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 53. Preferably, the PDGFRA protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 54.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is PDGFRB. Preferably, the PDGFRB protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 55. Preferably, the PDGFRB protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 56.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is selected from the group consisting of: FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is FZD1. Preferably, the FZD1 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 57. Preferably, the FZD1 protein is encoded by a sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 58.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is FZD2. Preferably, the FZD2 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 59. Preferably, the FZD2 protein is encoded by a sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 60.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is FZD3. Preferably, the FZD3 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 61. Preferably, the FZD3 protein is encoded by a sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 62.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is FZD4. Preferably, the FZD4 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 63. Preferably, the FZD4 protein is encoded by a sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 64.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is FZD5. Preferably, the FZD5 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 65. Preferably, the FZD5 protein is encoded by a sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 66.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is FZD6. Preferably, the FZD6 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 67. Preferably, the FZD6 protein is encoded by a sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 68.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is FZD7. Preferably, the FZD7 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 69. Preferably, the FZD7 protein is encoded by a sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 70.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is FZD8. Preferably, the FZD8 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 71. Preferably, the FZD8 protein is encoded by a sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 72.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is FZD9. Preferably, the FZD9 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 73. Preferably, the FZD9 protein is encoded by a sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 74.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is FZD10. Preferably, the FZD10 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 75. Preferably, the FZD10 protein is encoded by a sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 76.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is LRP5 or LRP6.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is LRP5. Preferably, the LRP5 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 77. Preferably, the LRP5 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 78.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is LRP6. Preferably, the LRP6 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 79. Preferably, the LRP6 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 80.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is a growth hormone antibody (GHR). Preferably, the GHR protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 84. Preferably, the GHR protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 85.

In a preferred embodiment, the transmembrane protein is used as an immune checkpoint inhibitor. Preferably, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is selected from the group consisting of PD-1, PD-L1, CTLA4, CMTM6, CMTM4 and WLS.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is PD-1. Preferably, the PD-1 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 86. Preferably, the PD-1 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 87.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is PD-L1. Preferably, the PD-L1 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 88. Preferably, the PD-L1 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 89.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is CTLA4. Preferably, the CTLA4 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 90. Preferably, the CTLA4 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 91.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is CMTM6. Preferably, the CMTM6 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 92. Preferably, the CMTM6 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 93.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is CMTM4. Preferably, the CMTM4 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 94. Preferably, the CMTM4 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 95.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is WLS/GPR177. Preferably, the WLS protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 100. Preferably, the WLS protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 101.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is SLC7A5. Preferably, the SLC7A5 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 102. Preferably, the SLC7A5 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 103.

In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is SLC16A7 (MCT 2). Preferably, the SLC16A7 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 104. Preferably, the SLC16A7 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 105.

Preferably, the second binding domain of the heterobifunctional molecule binds to an extracellular portion of a membrane-bound protein. Preferably, therefore, the heterobifunctional molecule does not have to cross the cell membrane to bind to the membrane-bound protein.

Preferably, the first binding domain and the second binding domain of the heterobifunctional molecule bind to the transmembrane E3 ubiquitin ligase and the extracellular portion of the membrane binding protein, respectively. Preferably, the heterobifunctional molecule binds extracellularly.

A first binding domain

The heterobifunctional molecules of the invention comprise at least a first binding domain and a second binding domain. The first binding domain is capable of specifically binding to a transmembrane E3 ubiquitin ligase. Preferably, the first binding domain is capable of specifically binding to a transmembrane E3 ubiquitin ligase as described in the section "protein bound by first binding domain" above. The first binding domain of the heterobifunctional molecule may bind selectively to a native epitope of the transmembrane E3 ubiquitin ligase, preferably when the heterobifunctional molecule is used in a medicament. Alternatively, the first binding domain of the heterobifunctional molecule may bind selectively to a non-native epitope engineered into a transmembrane E3 ubiquitin ligase, preferably when the heterobifunctional molecule is used in the method of the invention, preferably the selection method of the invention.

The first binding domain of the heterobifunctional molecule may be any domain capable of specifically binding to a transmembrane E3 ubiquitin ligase. Preferably, the first binding domain of the heterobifunctional molecule binds to the extracellular portion of a transmembrane E3 ubiquitin ligase.

The person skilled in the art will understand how to generate the first binding domain of the heterobifunctional molecules of the invention, e.g. by screening libraries of compounds, immunological studies and/or hybridoma techniques to generate antibodies or functional fragments thereof. Preferred functional antibody fragments are nanobodies. Details of these techniques are described, for example, in (Antibodies: A Laboratory Manual, harlow et al Cold Spring Harbor Publications, p.726, 1988), or by (Campbell, A.M. "Monoclonal Antibody Technology Techniques in Biochemistry and Molecular Biology," Elsevier Science Publishers, amsterdam, the Netherlands, 1984) or (St. Groth et al, J.Immunol. Methods 35:1-21,1980). Details of VHH/nanobody production against native epitopes are described, for example, in Pardon et al, nature Protocols 2014, which is incorporated herein by reference.

In a preferred embodiment, the molecule that binds to transmembrane E3 ubiquitin ligase is an antibody. Thus preferably, the antibody is useful as the first binding domain in a heterobifunctional molecule of the invention.

Preferably, the antibody is an antibody fragment. Preferably, the antibody fragment is a nanobody. Thus, in a preferred embodiment, the molecule that can bind to the transmembrane E3 ubiquitin ligase is a nanobody. Thus, preferably nanobodies can be used as the first binding domain in the heterobifunctional molecules of the invention.

In a preferred embodiment, the first binding domain is a small organic molecule.

In a preferred embodiment, the first binding domain is an aptamer.

In a preferred embodiment, the first binding domain is a protein molecule. The protein molecule may be cyclized, so preferably the protein molecule is a cyclic peptide. The peptide may be cyclized by direct covalent linkage between two amino acid residues or by the use of a crosslinking moiety. Such crosslinking moieties are well known in the art, such as, but not limited to, the crosslinking moieties described in WO2012/057624, which is incorporated herein by reference. The protein molecule may be a protein molecule known in the art.

Thus, the present invention extends to molecules known in the art that specifically bind to transmembrane E3 ubiquitin ligase, which can be used as the first binding domain of the heterobifunctional molecules of the invention. Such well-known molecules include, but are not limited to, at least one of well-known antibodies, protein molecules, aptamers, or well-known small organic molecules. Preferably, the antibody, protein molecule, aptamer or small organic molecule is well known in the art for binding to the extracellular portion of a transmembrane E3 ubiquitin ligase.

Antibodies that bind to transmembrane E3 ubiquitin ligase are well known in the art and can be readily recovered by those skilled in the art. Any known antibody capable of specifically binding to the transmembrane E3 ubiquitin ligase, preferably capable of specifically binding to the extracellular portion of the transmembrane E3 ubiquitin ligase, is suitable for use as the first binding domain in the heterobifunctional molecule of the invention.

A preferred known molecule that can bind to transmembrane E3 ubiquitin ligase is nanobody. Thus, preferably nanobodies can be used as the first binding domain in the heterobifunctional molecules of the invention.

In a preferred embodiment, the first binding domain is a native ligand of a transmembrane E3 ubiquitin ligase or a functional fragment thereof, i.e. a fragment of a native ligand capable of binding to a transmembrane E3 ubiquitin ligase.

As non-limiting examples, natural ligands for RNF43 and ZNRF3 are Rspondin (RSPO) -1, rspondin (RSPO) -2, rspondin (RSPO) -3 and Rspondin (RSPO) -4. Thus, in embodiments, the heterobifunctional molecule comprises a first binding domain capable of binding to RNF43, wherein the first binding domain is selected from the group consisting of: rspondin1, rspondin 2, rspondin 3 and Rspondin 4, or functional fragments thereof. Or, the heterobifunctional molecule comprises a first binding domain capable of binding to ZNRF3, wherein the first binding domain is selected from the group consisting of: rspondin1, rspondin 2, rspondin 3 and Rspondin 4, or functional fragments thereof.

A second binding domain

The heterobifunctional molecules of the invention comprise at least a first binding domain and a second binding domain. The second domain is capable of specifically binding to a membrane binding protein, preferably a transmembrane protein. Preferably, the second binding domain is capable of specifically binding to a membrane-bound protein as described in the section "protein bound by the second binding domain" above. The second binding domain of the heterobifunctional molecule can selectively bind to a native epitope of a membrane-bound protein, preferably when the heterobifunctional molecule is used in a medicament. Alternatively, the second binding domain of the heterobifunctional molecule may bind selectively to a non-native epitope engineered into a membrane-bound protein, preferably when the heterobifunctional molecule is used in the methods of the invention, preferably the selection methods of the invention.

The second binding domain of the heterobifunctional molecule may be any domain capable of specifically binding to a membrane binding protein, preferably a transmembrane protein. Preferably, the second binding domain of the heterobifunctional molecule binds to an extracellular portion of a membrane-bound protein.

The second binding domain may be an antibody, peptide, aptamer, or small organic molecule.

Those of skill in the art understand how to generate the second binding domain of the heterobifunctional molecules of the invention, e.g., by screening libraries of compounds, immunological studies, and/or hybridoma techniques to generate antibodies or functional fragments thereof. Preferred functional antibody fragments are nanobodies. Details of these techniques are described, for example, in (Antibodies: A Laboratory Manual, harlow et al Cold Spring Harbor Publications, p.726, 1988), or by (Campbell, A.M. "Monoclonal Antibody Technology Techniques in Biochemistry and Molecular Biology," Elsevier Science Publishers, amsterdam, the Netherlands, 1984) or (St. Groth et al, J.Immunol. Methods 35:1-21,1980).

In a preferred embodiment, the molecule that binds to the membrane-bound protein is an antibody. Thus, preferably, the antibody is useful as a second binding domain in a heterobifunctional molecule of the invention.

Preferably, the antibody is an antibody fragment. Preferably, the antibody fragment is a nanobody. Thus, in a preferred embodiment, the molecule that can bind to the membrane-bound protein is a nanobody. Preferably, nanobodies are therefore used as the second binding domain in the heterobifunctional molecules of the invention.

In a preferred embodiment, the first binding domain is an aptamer.

In a preferred embodiment, the second binding domain is a protein molecule. The protein molecule may be cyclized, so preferably the protein molecule is a cyclic peptide. The peptide may be cyclized by direct covalent linkage between two amino acid residues or by the use of a crosslinking moiety. Such crosslinking moieties are well known in the art, such as, but not limited to, the crosslinking moieties described in WO2012/057624, which is incorporated herein by reference. The protein molecule may be a protein molecule known in the art.

Thus, the present invention extends to molecules known in the art that specifically bind to membrane-bound proteins, preferably as defined above. Such molecules can be used as the second binding domain of the heterobifunctional molecules of the invention.

Such well-known molecules include, but are not limited to, at least one of well-known antibodies, protein molecules, aptamers, or well-known small organic molecules. Preferably, the antibody, protein molecule, aptamer or small organic molecule is well known in the art for binding to the extracellular portion of a membrane-bound protein as defined herein.

Antibodies that bind to membrane-bound proteins, preferably transmembrane proteins as defined above, are well known in the art and can be readily recovered by a person skilled in the art. Any known antibody capable of specifically binding to a membrane-binding protein as defined herein, preferably capable of specifically binding to an extracellular portion of a membrane-binding protein as defined herein, is suitable for use as the second binding domain in the heterobifunctional molecule of the invention.

Preferred known molecules that can bind to membrane-bound proteins are nanobodies. Thus, preferably nanobodies can be used as the second binding domain in the heterobifunctional molecules of the invention.

In a preferred embodiment, the second binding domain is a natural ligand of a membrane binding protein (preferably a transmembrane protein as defined herein). Preferably, the natural ligand is an antagonist of a transmembrane protein.

Heterobifunctional molecules

The first binding domain and the second binding domain are capable of specifically binding to a transmembrane protein or a membrane binding protein (i.e. a target protein), respectively.

In this context, "specific binding" is understood to mean that the domain of a "non-target" protein binds to less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the domain's binding to its specific target protein as determined by Fluorescence Activated Cell Sorting (FACS) analysis, ELISA or Radioimmunoassay (RIA). With respect to binding of a domain to a target protein, the term "specifically binds" or "specifically binds" to … or has "specificity" for a particular polypeptide or an epitope on a particular polypeptide target refers to binding that is significantly different from non-specific interactions. Specific binding can be measured, for example, by determining binding of a target protein as compared to binding of a control protein, which is typically a similar structural protein that does not have binding activity. For example, specific binding can be determined by competition with a control protein similar to the target protein, e.g., excess unlabeled target protein. In this case, specific binding is indicated if binding of the labeled target protein to the probe is competitively inhibited by an excess of unlabeled target protein.

The term "specific binding"Or "specifically bind to" … or "specifically bind to" a particular polypeptide or epitope on a particular polypeptide target as used herein can be accomplished, for example, by K against a target protein _d (as may be determined above) of at least about 10 ^-4 M, or at least about 10 ^-5 M, or at least about 10 ^-6 M, or at least about 10 ^-7 M, or at least about 10 ^-8 M, or at least about 10 ^-9 M, or at least about 10 ^- ¹⁰ M, or at least about 10 ^-11 M, or at least about 10 ^-12 M or larger binding domain. In one embodiment, the term "specifically binds" refers to a binding domain that binds to a particular polypeptide or an epitope on a particular polypeptide, but does not substantially bind to any other polypeptide or polypeptide epitope.

The heterobifunctional molecule, as a result of binding to both the transmembrane E3 ubiquitin ligase and the membrane bound protein, leads to ubiquitination and degradation of the transmembrane protein. Preferably, the membrane-bound protein is a transmembrane protein. Thus, preferably, the heterobifunctional molecule results in ubiquitination and degradation of the transmembrane protein as a result of binding to both the transmembrane E3 ubiquitin ligase and the transmembrane protein. Preferably, the degradation is at least one of proteasome degradation and lysosomal degradation. Preferably, the degradation is lysosomal degradation.

Thus, a heterobifunctional molecule as defined herein can knock-out or knock-out a membrane-bound protein present on a cell membrane by bringing the transmembrane E3 ubiquitin ligase into proximity of the target protein (i.e., membrane-bound protein). In other words, the steady state level of membrane-bound proteins will decrease.

Steady state levels may be defined herein as the abundance of protein in each cell. The steady state level of the membrane-bound protein may be reduced by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or may be reduced by about 100% compared to a reference cell, i.e., binding to the heterobifunctional molecule results in complete deletion of the membrane-bound protein.

In a preferred embodiment, the heterobifunctional molecule is a bispecific antibody. Bispecific antibodies are described, for example, in Wu Xand Demarest SJ, methods (2019) 154:3-9. Thus, preferably, the first binding domain is an antibody capable of specifically binding to a transmembrane E3 ubiquitin ligase (preferably a transmembrane E3 ubiquitin ligase as described in the section "transmembrane E3 ubiquitin ligase bound by first binding domain" above). Preferably, the second binding domain is also an antibody, wherein the antibody is capable of specifically binding to a membrane binding protein (preferably a transmembrane protein, preferably a transmembrane protein as described in the section "protein bound by the second binding domain" above). The two antibodies (i.e., the first binding domain and the second binding domain) may be coupled together directly, or there may be a linker between the two antibodies, preferably a linker as described herein.

In a preferred embodiment, the heterobifunctional molecule is a bispecific nanobody. Bispecific nanobodies are disclosed, for example, in WO 2015/044386 and Conrath et al (Camel Single-domain Antibodies as Modular Building Units inBispecific and Bivalent Antibody Constructs, JBC, 2001). Preferably, the first binding domain is a nanobody capable of specifically binding to a transmembrane E3 ubiquitin ligase (preferably a transmembrane E3 ubiquitin ligase as described in the section "transmembrane E3 ubiquitin ligase bound by first binding domain" above). Preferably, the second binding domain is also a nanobody, wherein the nanobody is capable of specifically binding to a membrane binding protein (preferably a transmembrane protein, preferably a transmembrane protein as described in the section "protein bound by the second binding domain" above). The two nanobodies (i.e., the first binding domain and the second binding domain) may be coupled together directly, or there may be a linker between the two nanobodies, preferably a linker as described herein.

In a preferred embodiment, the heterobifunctional molecule is a bicyclic peptide. Preferably, the first binding domain is a cyclic peptide capable of specifically binding to a transmembrane E3 ubiquitin ligase (preferably a transmembrane E3 ubiquitin ligase as described in the section "transmembrane E3 ubiquitin ligase bound by first binding domain" above). Preferably, the second binding domain is also a cyclic peptide, wherein the cyclic peptide is capable of specifically binding to a membrane binding protein (preferably a transmembrane protein, preferably a transmembrane protein as described in the section "protein bound by the second binding domain" above). The two cyclic peptides (i.e., the first binding domain and the second binding domain) can be coupled together directly (e.g., by using the same cross-linking moiety), or there can be a linker between the two cyclic peptides, preferably a linker as described herein.

The heterobifunctional molecule may optionally comprise a moiety that enhances the stability of the molecule. Such moieties include, but are not limited to, binding domains for specifically binding albumin.

The heterobifunctional molecule may optionally comprise a tag, preferably a peptide tag or a protein tag as defined herein, for purifying or detecting the heterobifunctional molecule. Preferred purification tags are His tags or Avi tags. The preferred detection tag is a V5 tag. Such heterobifunctional molecules are particularly suitable for use in the (screening) methods of the invention.

The heterobifunctional molecules of the invention may comprise a linker between the first binding domain and the second binding domain. The linker may be any suitable linker known in the art. Preferably, the linker is a Gly-Ser sequence. One skilled in the art knows how to select a linker based on a first binding domain and a second binding domain. The linker may be a very flexible linker, for example in the form of (GGGGS) n, (GGS) n and (G) n, to a more rigid linker in the form of (EAAAK) n, (SPKKKRKVEAS) n (SEQ ID NO: 81) or (SGSETPGTSESATPES) n (SEQ ID NO: 82) or (KSGSETPGTSESATPES) n (SEQ ID NO: 83), or any variant thereof, wherein n is 1 to 15, preferably 1 to 7, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

The linker preferably has a length of 2 to 250 amino acids, a length of 2 to 30 amino acids, or a length of 3 to 23 amino acids, or a length of 3 to 18 amino acids.

The linker may be a cleavable linker, for example by introducing a 3C protease cleavage site in the linker. Such a linker is particularly useful in the (screening) method of the invention, as it provides a control, e.g. by cleavage of the linker, forced dimerization can be eliminated.

Therapeutic use

The heterobifunctional molecules defined herein can be used to reduce the level of a selected membrane-bound protein by binding to both the transmembrane ubiquitin E3 ligase and any selected membrane-bound protein. The combination of transmembrane ubiquitin ligase and the selected membrane-bound protein is preferably an effective combination, preferably a combination selected using the selection method of the invention.

In one aspect, the heterobifunctional molecule as defined herein is for use as a medicament. The heterobifunctional molecule for use as a medicament preferably binds to a native epitope present on the transmembrane ubiquitin E3 ligase and a native epitope present on the selected membrane bound protein. The medical use described herein is expressed as a heterobifunctional molecule as defined herein being used as a medicament for treating said disease by administering an effective amount of the heterobifunctional molecule, but can equally be expressed as a method of treating said disease using a heterobifunctional molecule as defined herein, the method comprising the step of administering to a subject an effective amount of the heterobifunctional molecule, a heterobifunctional molecule as defined herein for the preparation of a medicament for treating said disease, wherein the heterobifunctional molecule is administered in an effective amount, and treating said disease with the heterobifunctional molecule as defined herein by administering an effective amount. Such medical uses are contemplated by the present invention.

It will be appreciated by those skilled in the art that any increase in the activity of a membrane-bound protein involved in the onset, severity or duration of a disease may be a suitable target for the heterobifunctional molecule defined herein. Thus, the heterobifunctional molecule is not limited to any particular membrane-bound protein or any particular disease. Preferably, the disease is characterized by an increase in the activity of a membrane-bound protein (preferably a receptor), wherein the increase in the activity of the membrane-bound receptor preferably affects or dominates the onset, severity or duration of the disease.

As a non-limiting example, the heterobifunctional molecule may be used to treat at least one of cancer, dementia, heart disease, neurological disorders, rare diseases, and infectious diseases.

The increased activity of membrane-bound receptors plays an important role in, for example, the onset, severity or duration of cancer, as is well known in the art. Thus, in embodiments, the heterobifunctional molecules are useful for treating, preventing, reducing, or inhibiting symptoms associated with cancer.

Preferably, the cancer is a cancer as defined in the section "protein bound by the second binding domain" above.

Preferably, the cancer is a solid cancer or a hematological cancer. Alternatively or in addition, the receptor may be involved in cancer angiogenesis.

Preferably, the solid cancer is a solid cancer as defined in the section "protein bound by the second binding domain" above.

Preferably, the hematological cancer is a hematological cancer as defined in the section "protein bound by the second binding domain" above.

In one aspect, the present invention relates to a composition comprising a heterobifunctional molecule as defined herein. The heterobifunctional molecule preferably binds to an effective combination of a transmembrane ubiquitin E3 ligase and a membrane-bound protein, preferably selected using the selection method of the invention. The composition is suitable for cell culture, preferably animal cell culture, more preferably mammalian cell culture. Additionally or alternatively, the composition is preferably a pharmaceutical or cosmetic composition.

The composition may comprise one type of heterobifunctional molecule, or may comprise at least two different types of heterobifunctional molecules, for example to knock-out or knock-out the presence of two or more different membrane-bound proteins. The composition may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different types of heterobifunctional molecules.

Compositions comprising the heterobifunctional molecules as described above may be prepared as pharmaceutical or cosmetic formulations, or in various other media, such as human or animal food products, including medical foods and dietary supplements.

A "medical food" is a product intended for specific dietary management of a disease or condition for which special nutritional needs exist. By way of example, but not limitation, medical foods may include vitamin and mineral formulations that are fed through a feeding tube (known as enteral administration).

"dietary supplement" refers to a product intended to supplement the human diet, typically provided in the form of a formulation such as a pill, capsule, tablet, or the like. For example, but not limiting of, a dietary supplement may comprise one or more of the following: vitamins, minerals, herbs and botanicals; amino acids, dietary substances intended to supplement the diet by increasing the total dietary intake, concentrates, metabolites, ingredients, extracts or combinations of any of the above. Dietary supplements may also be added to foods including, but not limited to, food bars, beverages, powders, cereals, delicatessens, food additives, and confectioneries.

Thus, the compositions of the present invention may be mixed with other physiologically acceptable materials that may be ingested, including but not limited to food products. Additionally or alternatively, the compositions described herein may be administered orally in combination with (separate) consumption of food.

The composition may be administered alone or in combination with other pharmaceutical or cosmetic agents, and may be combined with a physiologically acceptable carrier therefor. In particular, the heterobifunctional molecules described herein can be formulated into pharmaceutical or cosmetic compositions by being formulated with additives (e.g., pharmaceutically or physiologically acceptable excipient carriers and vehicles).

Suitable pharmaceutically or physiologically acceptable excipients, carriers, and vehicles include processing agents and drug delivery modifiers and enhancers, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, glucose, hydroxypropyl-P-cyclodextrin, polyvinylpyrrolidone, low melting waxes, ion exchange resins, and the like, as well as combinations of any two or more thereof. Other suitable pharmaceutically acceptable excipients are described in "Remington's Pharmaceutical Sciences," Mack pub.co., new Jersey (1991), and "Remington: the Science and Practice of Pharmacy," Lippincott Williams&Wilkins,Philadelphia,20 ^th edition(2003),21 ^st edition(2005)and 22 ^nd edition (2012), which is incorporated herein by reference.

The pharmaceutical or cosmetic composition comprising the heterobifunctional molecule used according to the invention may be in any form suitable for the intended method of administration, including for example solutions, suspensions or emulsions. In a preferred embodiment, the heterobifunctional molecule is administered in solid form or in liquid form.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders and granules. In such solid dosage forms, the heterobifunctional molecule may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also contain other substances in addition to the inert diluent, for example, a lubricant, such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills may also be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art (e.g., water or saline). Such compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents.

Liquid carriers are commonly used in the preparation of solutions, suspensions and emulsions. In preferred embodiments, liquid carriers/liquid dosage forms contemplated for use in the practice of the invention include, for example, water, saline, pharmaceutically acceptable organic solvents, pharmaceutically acceptable oils or fats, and the like, as well as mixtures of two or more thereof. In a preferred embodiment, the heterobifunctional molecule of the invention as defined herein is mixed with an aqueous solution prior to administration. The aqueous solution should be suitable for application and such aqueous solutions are well known in the art. It is also well known in the art that the suitability of an aqueous solution for administration may depend on the route of administration.

In a preferred embodiment, the aqueous solution is an isotonic aqueous solution. The aqueous isotonic solution is preferably nearly (or completely) isotonic with blood plasma. In an even more preferred embodiment, the aqueous solution is saline.

The liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity modifiers, stabilizers, flavoring agents and the like. Preferred flavoring agents are sweeteners, such as mono-and/or disaccharides. Suitable organic solvents include, for example, monohydric alcohols (e.g., ethanol) and polyhydric alcohols (e.g., glycols). Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like.

For parenteral administration, the carrier may also be an oily ester of ethyl oleate, isopropyl myristate, or the like. The compositions for use in the present invention may also be in the form of microparticles, microcapsules, liposome encapsulates, and the like, as well as combinations of any two or more thereof.

Time release, slow release or controlled release delivery systems, such as diffusion controlled matrix systems or erodible systems, may be used, as described below: lee, "Diffusion-Controlled Matrix Systems", pp.155-198and Ron and Langer, "Erodible Systems", pp.199-224,in"Treatise on Controlled Drug Delivery", A.Kydonieus Ed., marcel Dekker, inc., new York 1992. The matrix may be, for example, a biodegradable material that can spontaneously degrade in situ and in vivo, e.g., by hydrolysis or enzymatic cleavage, such as by proteases. The delivery system may be, for example, a natural or synthetic polymer or copolymer, such as in the form of a hydrogel. Exemplary polymers having cleavable linkages include polyesters, polyorthoesters, polyanhydrides, polysaccharides, poly (phosphates), polyamides, polyurethanes, poly (iminocarbonates), and poly (phosphazenes).

The heterobifunctional molecules of the invention may also be administered in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances, as is well known in the art. Liposomes are formed from a single or multiple layers of hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The compositions of the present invention in liposome form may contain stabilizers, preservatives, excipients, and the like, in addition to the heterobifunctional molecules defined herein. Preferred lipids are natural and synthetic phospholipids and phosphatidylcholines (lecithins). Methods of forming liposomes are well known in the art. See, e.g., prescott, ed., methods in Cell Biology, volume XIV, academic Press, new York, n.y., p.33et seq (1976).

The pharmaceutical or cosmetic composition may comprise a unit dose formulation, wherein the unit dose is a dose sufficient to produce a therapeutic or inhibitory effect on a disease or disorder as defined herein, and/or an amount effective to reduce or knock-out expression of the membrane-associated protein. The unit dose may be sufficient as a single dose having a therapeutic or inhibitory effect on a disease or condition as defined herein, and/or an amount effective to reduce expression of the target membrane-associated protein. Alternatively, a unit dose may be a dose that is periodically administered during the treatment or inhibition of a disease or disorder as defined herein. The concentration of the test composition may be monitored during the course of treatment to ensure that the desired level is maintained.

The heterobifunctional molecule or composition comprising the heterobifunctional molecule as defined herein may be administered enterally, orally, parenterally, sublingually, by inhalation (e.g., vapor or spray), rectally, or topically, preferably in dosage unit formulations containing conventional non-toxic, pharmaceutically or physiologically acceptable carriers, adjuvants, and vehicles as desired. For example, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoretic, intravenous, intraarterial, intramuscular, intraperitoneal, intranasal (e.g., through the nasal mucosa), subdural, rectal, gastrointestinal, and the like, as well as direct administration to a particular or affected organ or tissue (e.g., cancerous tissue). For delivery to the central nervous system, spinal and epidural administration may be used, or to the ventricle. Topical administration may also include use of transdermal administration, such as transdermal patches or iontophoresis devices. The term parenteral administration as used herein includes subcutaneous injections, intravenous injection, intramuscular injection, intrasternal injection or infusion techniques.

The heterobifunctional molecule may be admixed with pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for the desired route of administration. The heterobifunctional molecules of the invention may be administered by transgastric or percutaneous tube supplementation.

In a preferred embodiment, the present invention relates to a heterobifunctional molecule as described above for treating, preventing or inhibiting a symptom associated with cancer by administering an effective total daily dose.

The dosage form for oral administration may be a solid oral dosage form. The class of solid oral dosage forms consists primarily of tablets and capsules, but other dosage forms are well known in the art and are equally suitable. When used as a solid oral dosage form, the heterobifunctional molecule as defined herein may be administered, for example, in the form of an immediate release tablet (or capsule, etc.) or a sustained release tablet (or capsule, etc.). Any suitable immediate or sustained release solid dosage form may be used in the context of the present invention, as will be apparent to those skilled in the art.

The heterobifunctional molecule as used herein may be administered in solid form, liquid form, aerosol form, or in tablet, pill, powder mixture, capsule, granule, injection, cream, solution, suppository, enema, colon rinse, emulsion, dispersion, food premix, and other suitable forms. The heterobifunctional molecule may also be administered in a liposomal formulation. The compounds may also be administered as prodrugs, wherein the prodrugs are converted to a therapeutically effective form in the subject receiving the treatment. Other modes of administration are also well known in the art.

Injectable formulations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in propylene glycol. Acceptable carriers and solvents that can be employed include water, ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids (e.g., oleic acid) find use in the preparation of injectables.

Suppositories for rectal administration of the heterobifunctional molecule can be prepared by mixing the heterobifunctional molecule with suitable non-irritating excipients (e.g. cocoa butter and polyethylene glycols) which are solid at room temperature and liquid at the rectal temperature and therefore melt in the rectum and release the heterobifunctional molecule.

Although the heterobifunctional molecules used as described herein may be administered as the sole active pharmaceutical (or cosmetic) agent, they may also be used in combination with one or more other agents for treating or inhibiting a disease or disorder. Representative agents that may be used in combination with the heterobifunctional molecules of the present invention to treat, prevent, or inhibit symptoms associated with a disease or disorder include, but are not limited to, coenzyme Q, vitamin E, idebenone, mitoQ, EPI-743, vitamin K and its analogs, naphthoquinone and its derivatives, other vitamins, and antioxidant compounds.

When other active agents are used in combination with the heterobifunctional molecules of the present invention, the other active agents may generally be used in accordance with the therapeutic amounts described in the Physics' Desk Reference (PDR) 53rd Edition (1999), which is incorporated herein by Reference, or in therapeutically useful amounts known to those of ordinary skill in the art. The heterobifunctional molecules of the invention and other therapeutically active agent(s) may be administered at the recommended maximum clinical dose or at lower doses. The dosage level of the active compound in the compositions of the present invention may vary depending on the route of administration, the severity of the disease and the patient's response to achieve the desired therapeutic response. When administered in combination with other therapeutic agents, the therapeutic agents may be formulated as separate compositions for simultaneous or non-simultaneous administration, or the therapeutic agents may be administered as a single composition.

Preparation of heterobifunctional molecules

In one aspect, the present invention relates to a method for preparing a heterobifunctional molecule of the invention, wherein the method comprises the steps of:

-selecting an effective combination of a transmembrane E3 ubiquitin ligase and a membrane binding protein, preferably using the method of the invention;

-constructing a first binding domain capable of specifically binding to the transmembrane E3 ubiquitin ligase;

-constructing a second binding domain capable of specifically binding to the membrane-bound protein; and

coupling the first binding domain to the second binding domain, wherein preferably the coupling is direct coupling or coupling via a linker, preferably a linker as defined herein.

It will be appreciated herein that the step of constructing the first binding domain and the second binding domain may be performed using any means conventional in the art. As a non-limiting example, at least one of the first binding domain and the second binding domain is a binding domain previously known in the art, e.g., an antibody known in the art for specifically binding to transmembrane E3 ubiquitin ligase or an antibody known in the art for specifically binding to a membrane bound protein.

Or, at least one of the first binding domain and the second binding domain is a de novo binding domain, such as, but not limited to, an antibody or nanobody binding domain found in immunoresearch.

The step of selecting a transmembrane E3 ubiquitin ligase and a membrane bound protein can be accomplished by combining a first non-native epitope tag with the transmembrane E3 ubiquitin ligase and combining a second non-native epitope tag with the membrane bound protein. After expression in the cell, the first epitope tag and the second epitope tag are preferably displayed in the respective extracellular domains, i.e. extracellularly. Subsequently, heterobifunctional molecules having a first binding domain capable of binding to a first epitope tag and a second binding domain capable of binding to a second epitope tag can be used to assess the efficacy of targeting transmembrane E3 ubiquitin ligase to a membrane-bound protein. The efficacy can be assessed by determining to what extent a transmembrane E3 ubiquitin ligase is able to ubiquitinate and internalize a membrane bound protein following forced interaction between the E3 ubiquitin ligase and the membrane bound protein using a heterobifunctional molecule. Additionally or alternatively, the efficacy can be assessed by determining to what extent the cell surface level and total protein level of the membrane bound protein is reduced following forced interaction between the E3 ubiquitin ligase and the membrane bound protein using the selection system. The step of selecting a transmembrane E3 ubiquitin ligase and a membrane-binding protein can also be considered as a method for reducing the surface level of a cell membrane-binding protein as detailed below.

After selection of an effective combination of transmembrane E3 ubiquitin ligase and membrane binding protein, a heterobifunctional molecule comprising a first binding domain capable of specifically binding to the extracellular portion of the (native) transmembrane E3 ubiquitin ligase and a second binding domain capable of specifically binding to the extracellular portion of the (native) membrane binding protein can be constructed.

Drawings

FIG. 1.Schematic of an exemplary embodiment of the present invention. The heterobifunctional molecules of the invention bind both transmembrane E3 ubiquitin ligase and transmembrane proteins. Thus, transmembrane proteins will ubiquitinate, internalize and degrade.

Fig. 2.Functional assessment of A/C dimer. HEK293T cells were transfected with RNF43-FKBP and T beta RII-Flag-FRB and treated overnight with A/C dimer or similar volumes of 100% ethanol. The T.beta.RII construct was immunoprecipitated from cell lysates using Flag-M2 magnetic beads. IP samples and whole cell lysates were separated by SDS-page, western blots and staining were performed for Flag and RNF43 to detect binding between the two constructs.

Fig. 3.Forced dimerization of RNF43 and tbrii induces the relocation of both proteins to the nuclear Zhou Rong enzyme. (a) Confocal images of HEK293T cells transfected with tbrii-Flag-FRB. (B) Confocal images of HEK293T cells transfected with RNF43-FKBP and T beta RII-Flag-FRB. Cells were treated with a/C dimer or similar volumes of 100% ethanol overnight. T beta RII and RNF43 were shown by Flag and RNF43 staining, respectively. (C) Confocal images of HEK293T cells transfected with CD63-GFP, RNF43-FKBP and T beta RII-Flag-FRB. Cells were treated with A/C dimer overnight and showed T.beta.RII-Flag-FRB by Flag staining. The arrow indicates the core Zhou Rong enzyme.

Fig. 4.Tβrii is degraded after RNF43 and tβrii have forced dimerization. HEK293T cells were transfected with RNF43-FKBP and T beta RII-Flag-FRB and treated overnight with A/C dimer or similar volumes of 100% ethanol. Cell lysates were separated by SDS-page, western blotted and stained for Flag and RNF43 to show protein levels.

Fig. 5.VHH-mediated dimerization of RNF43 or RNF167 with the receptor tbrii or EGFR induces receptor internalization and copolymerization into the perinuclear region. Prior to fixing byCells were treated with 100nM bi-VHH for 5 hours. The E3 ligase was shown by Myc staining and the receptor by Flag staining. (A) Confocal images of HEK293T cells transfected with E6-Flag-T beta RII and Alpha-Myc-RNF 43. (B) Confocal images of HEK293T cells transfected with E6-Flag-T beta RII and Alpha-Myc-RNF 167. (C) Confocal images of HEK293T cells transfected with E6-Flag-EGFR and Alpha-Myc-RNF 43. (D) Confocal images of HEK293T cells transfected with E6-Flag-EGFR and Alpha-Myc-RNF 167. Arrows indicate co-aggregation of E3 ligase and receptor in the perinuclear region.

Fig. 6.Bifunctional VHH treatment facilitates membrane-bound E3 ligase-mediated internalization of transmembrane receptors from the cell surface. HEK293T cells were transfected with at least one of the E3 ligases RNF43, RNF128, RNF130 or RNF167 and the receptors CTLA-4, FLT-3, PD-1 and PD-L1. Cells were kept untreated or treated with 50nM bi-VHH overnight prior to fixation. Receptors present on the cell surface are shown by Flag staining of the non-permeabilized cells. (A) Confocal images of HEK293T cells transfected with E6-Flag-CTLA-4 and Alpha-Myc-RNF43 or Alpha-Myc-RNF 167. (B) Confocal images of HEK293T cells transfected with E6-Flag-FLT-3 and Myc-RNF43, alpha-Myc-RNF128 or Alpha-Myc-RNF 167. (C) Confocal images of HEK293T cells transfected with E6-Flag-PD-1 and Alpha-Myc-RNF43, alpha-Myc-RNF128, alpha-Myc-RNF130 or Alpha-Myc-RNF 167. (D) Confocal images of HEK293T cells transfected with E6-Flag-PD-L1 and Alpha-Myc-RNF43, alpha-Myc-RNF128, alpha-Myc-RNF130 or Alpha-Myc-RNF 167.

Fig. 7.Bifunctional VHH treatment promotes membrane-bound E3 ligase-mediated internalization of type III multi-transmembrane protein CMTM6 from the cell surface. (A) Schematic representation of Snorkel tags for detecting surface expression of multi-spanning proteins. (B) HEK293T cells were transfected with one of the E3 ligases RNF43, RNF128, RNF130 or RNF167 and the multi-transmembrane receptor CMTM 6. Cells were kept untreated or treated with 50nM bi-VHH overnight prior to fixation. Receptors present on the cell surface are shown by Flag staining of the non-permeabilized cells. Confocal images of HEK293T cells transfected with E6-Flag-Snorkel-CMTM6 and Alpha-Myc-RNF43, alpha-Myc-RNF128, alpha-Myc-RNF130 or Alpha-Myc-RNF167 are shown.

FIG. 8 shows the effect of bi-VHH on the validation of E3 ligase target combination at endogenous level. (A) strategy for the production of endogenous marker proteins. (B) Schematic of a method for removal of target protein cell surface by bi-VHH using an endogenous, labeled version of the E3 ligase-target protein combination.

Examples

Example 1

Materials and methods

Cell culture and transfection

Human Embryonic Kidney (HEK) 293T cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (GE Healthcare), 2mM UltraGlutamine (Lonza), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen). Cells were incubated at 37℃with 5% CO ₂ Is cultured. Transfection was performed using FuGENE 6 (Promega) or biochemical procedures using PEI according to the manufacturer's microscopy protocol. A/C heterodimer (Takara Bio, # 635056) was used overnight at 37℃in an amount of 1uM, and the control conditions were treated with an equal amount of 100% ethanol. TGF-beta was used at a concentration of 1.5ng/mL for 45 minutes.

Constructs and antibodies

Type II TGF-beta serine/threonine kinase receptors (T beta RII) -Flag-FKBP and (T beta RII) -Flag-FRB are provided by Peter ten Dijke (LUMC, leiden). RNF43-FKBP and RNF43-FRB were prepared by mixing FKBP using a Q5 High-Fidelity 2x master mix (Q5 High-Fidelity 2x Master Mix,NEB), respectively ^36V Or FRB, into the C-tail of human RNF 43. All constructs were sequence verified. CD63-GFP was from J.Klumpeerman (UMCU, utrecht). The following primary antibodies were used for Immunoblotting (IB), immunofluorescence (IF) or Immunoprecipitation (IP): rabbit anti-FLAG (Sigma-Aldrich), rat anti-HA (Roche), mouse anti-FLAG (M2; sigma-Aldrich), mouse anti-Actin (MP Biomedicals) and rabbit anti-RNF 43 (Sigma-Aldrich). These primary antibodies were diluted according to the manufacturer's instructions. Secondary antibodies for IB or IF were used at 1:8000 or 1:300 respectively and were obtained from Rockland or Invitrogen.

Immunofluorescence and confocal microscopy. HEK293T cells were grown on laminin (Sigma) coated glass coverslips in 24 well plates. After overnight transfection, cells were fixed in Phosphate Buffered Saline (PBS) with 4% formaldehyde. Cells were blocked in PBS buffer containing 2% bsa and 0.1% saponin for 30 min at Room Temperature (RT). Subsequently, cells were incubated with primary and secondary antibodies in blocking buffer for 1 hour at RT. Cells were mounted in Prolong Diamond (Life technologies) and images were acquired with an LSM700 confocal microscope. The images were analyzed and processed with ImageJ.

Immunoprecipitation and western immunoblotting. Following transfection, cells were grown to 80% confluency in 10cm dishes. The cells were washed with PBS, then scraped and washed with a solution containing 100mM NaCL, 50mM Tris pH 7.5, 0.25% Triton X-100, 10% Glycerol (10% glycerol), 50mM NaF, 10mM Na ₃ VO ₄ Lysis in cell lysis buffer of 10. Mu.M leupeptin, 10. Mu.M aprotinin and 1mM PMSF. Lysates were removed after centrifugation at 16.000Xg for 15 min at 4 ℃. Lysates were placed in SDS sample buffer and heated at 37℃for 1 hour. For immunoprecipitation, lysates were incubated with 25. Mu.l of pre-coupled Flag-M2 magnetic beads (Sigma) at 4℃and overnight. After washing, the beads were eluted with sample buffer and heated at 37 ℃ for 1 hour. After SDS-PAGE, proteins were transferred by western immunoblotting onto Immobilon-FL PVDF membrane (Miltiore). Blocking was performed with Odyssey blocking buffer (LI-COR) and then proteins were labeled with the indicated primary antibodies detected with goat anti-mouse/rabbit Alexa 680 (Invitrogen), donkey anti-mouse Alexa 680 (Invitrogen) or goat anti-mouse/rabbit IRDye 800 (Rockland) using an Amersham Typhoon biomolecular imager (GE Health Care).

Results and discussion

Forced dimerization of RNF43 and type II TGF- β serine/threonine kinase receptor (tbrii) induces lysosomal localization and degradation of tbrii

To demonstrate the concept of transmembrane E3 ligase redirection to target selected cell surface proteins for internalization and lysosomal degradation, we used the FKBP/FRB dimerization system. We fused the FKBP domain or FRB domain to the C-terminal tail of tbrii and RNF 43. When co-expressed in HEK293T cells, these proteins did not interact (fig. 2). However, addition of A/C dimer induced co-immunoprecipitation of RNF43 and T beta RII (FIG. 2). The dimer itself did not interfere with the stability of either tβrii or RNF43 (fig. 2, whole cell lysate). Next, we explored whether the forcible interaction of RNF43 with tβrii altered subcellular localization of tβrii. Regardless of whether RNF43 is absent (fig. 3A) or co-expressed (fig. 3B), tβrii is primarily localized on the plasma membrane in the absence of dimers. However, the addition of dimer strongly induced the relocation of tβrii and RNF43 to perinuclear vesicle clusters positive for lysosomal marker CD63 (fig. 3C). These findings indicate that forced dimerization of RNF43 and tbrii increases tbrii-directed lysosomes levels.

To determine if enhanced lysosomal localization of tβrii would result in a decrease in functional tβrii, we analyzed protein levels using western blotting. Although RNF43 expression itself had no effect on the stability of tbrii, induced RNF43 and tbrii dimerization clearly resulted in reduced tbrii protein levels (fig. 4). These results indicate that forced dimerization of the transmembrane E3 ligase RNF43 and the normally unrelated transmembrane receptor tβrii targets tβrii for lysosomal degradation.

Example 2

Materials and methods

Cell culture and transfection

Human Embryonic Kidney (HEK) 293T cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (GE Healthcare), 2mM UltraGlutamine (Lonza), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen). Cells were incubated at 37℃with 5% CO ₂ Is cultured. Transfection was performed using FuGENE 6 (Promega) according to the manufacturer's protocol.

Constructs and antibodies

Type II E6-Flag-TGF-beta serine/threonine kinase receptor (TβRII) and-Epidermal Growth Factor Receptor (EGFR) and Alpha-myc-RNF43 and RNF167 were obtained by subcloning using Q5 High-Fidelity 2x Master Mix (NEB). All constructs were sequence verified. The following primary antibodies were used for Immunofluorescence (IF): rabbit anti-Flag (Sigma-Aldrich) and mouse anti-Myc (hybrid 9E 10). These primary antibodies were diluted according to the manufacturer's instructions. The secondary antibody for IF was used at 1:300 (Life technologies).

Immunofluorescence and confocal microscopy. HEK293T cells were grown on laminin (Sigma-Aldrich) coated glass coverslips in 24-well plates. After overnight transfection, cells were incubated with 20nM Bafilomycin A1 (bafilomycin A1, sigma-Aldrich) for 1 hour before and during 5 hours of treatment with 100nM bi-VHH (VHH Alpha- (G4S) 3-VHH E6). After treatment, the cells were washed twice with warm medium and fixed in 0.05M phosphate buffer (pH 7.4) in 4% formaldehyde. Cells were blocked in PBS buffer containing 2% bsa and 0.1% saponin for 30 min at Room Temperature (RT). Subsequently, cells were incubated with primary antibodies against Flag or Myc for 1 hour at RT, followed by incubation with secondary antibodies in blocking buffer for 1 hour at RT. Cells were mounted in Prolong Diamond (Life technologies) and images were acquired with an LSM700 confocal microscope. The images were analyzed and processed with ImageJ.

Results and discussion

RNF43 and RNF167 induce cell surface depletion of tbrii and EGFR upon forced dimerization using bispecific VHH.

To further confirm the functionality of the heterobifunctional molecules of the invention, selected receptors were targeted with E3 ligase by VHH mediated dimerization of extracellular regions. To this end, we fused the epitope tag to the extracellular domains of the target protein (E6 tag) and the E3 ligase (Alpha tag). We selected these epitope tags for VHH recognition et al 2019,Nature Communications,10 (1), 1-12; ling et al 2019,Molecular Immunology,114 (July), 513-523), and we generated bispecific VHH (bi-VHH) against both epitopes to allow VHH mediated dimerization. To determine changes in protein localization, we also incorporated Myc epitope tags for the E3 ligase and Flag epitope tags for the receptor. None of them was found to be co-expressed in HEK293T cellsReceptor co-localization with any E3 ligase: the E3 ligase is mainly localized to the intracellular compartment, while the receptor is mainly localized to the plasma membrane (data not shown). However, after 5 hours of treatment with bi-VHH, both RNF43 and RNF167 induced removal of tbrii and EGFR from plasma membranes. Furthermore, in bafilomycin-treated cells, internalized proteins co-aggregated in the perinuclear region, which inhibited lysosomal conversion, suggesting that E3 ligase and its target protein accumulate in late endosomal/lysosomal structures (fig. 5A-5D). These findings indicate that the heterobifunctional molecule (e.g., bi-VHH) can be used to deliberately dimerize a transmembrane E3 ligase with a selected transmembrane receptor, thereby inducing receptor removal from the cell surface.

Example 3

Materials and methods

Cell culture and transfection

Human Embryonic Kidney (HEK) 293T cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (GE Healthcare), 2mM UltraGlutamine (Lonza), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen). Cells were incubated at 37℃with 5% CO ₂ Is cultured. Transfection was performed using FuGENE 6 (Promega) or Effectene (Qiagen) according to the manufacturer's protocol.

Constructs and antibodies

E6-Flag-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), receptor tyrosine protein kinase receptor FLT3 (FLT-3), apoptosis protein 1 (PD-1) and apoptosis protein 1 ligand 1 (PD-L1) and Alpha-myc-RNF43, RNF128, RNF130 and RNF167 were obtained by subcloning using Q5 High-Fidelity 2x Master Mix (NEB). All constructs were sequence verified. The following primary antibodies were used for Immunofluorescence (IF): rabbit anti-Flag or mouse anti-Flag (Sigma-Aldrich). These primary antibodies were diluted according to the manufacturer's instructions. The secondary antibody for IF was used at 1:300 (Life technologies).

Immunofluorescence and confocal microscopy. HEK293T cells were grown on laminin (Sigma-Aldrich) coated glass coverslips in 24-well plates. Six hours after transfection, cells were incubated overnight with 50nM bi-VHHs (VHH Alpha- (G4S) 3-VHH E6). After treatment, the cells were washed twice with warm medium and fixed in a solution containing 4% formaldehyde in 0.05M phosphate buffer (pH 7.4). Cells were blocked in PBS buffer containing 2% bsa for 30 min at Room Temperature (RT). Subsequently, cells were incubated with primary antibodies against Flag for 1 hour at RT, followed by incubation with secondary antibodies in blocking buffer for 1 hour at RT. Cells were mounted in Prolong Diamond (Life technologies) and images were acquired with LSM700 confocal microscope using 5-fold objective or with EVOS-M5000 microscope using 20-fold objective. The images were analyzed and processed with ImageJ.

Results and discussion

The specific E3 ligase-target protein combination allows removal of target protein from the surface upon forced dimerization using bispecific VHH.

To screen for other candidate E3 ligase-receptor combinations, we generated in addition to the previously generated constructs the following constructs: alpha-Myc-RNF128, alpha-Mcy-RNF130, E6-Flag-CTLA-4, E6-Flag-FLT-3, E6-Flag-PD-1 and E6-Flag-PD-L1. When co-expressed in HEK293T cells, CTLA-4, FLT-3, PD-1 and PD-L1 are all localized on the cell surface. Following overnight treatment with bi-VHH, the following E3-target protein combinations resulted in removal of target protein from the surface: CTLA-4 and RNF167; FLT-3 and RNF43, RNF128 or 167, PD-1 and RNF128, RNF130 or 167 and PD-L1 and RNF43, RNF128 or RNF130 (FIGS. 6A-6D). These findings expand the scope of use of heterobifunctional molecules (e.g., bi-VHHs) to deliberately dimerize various transmembrane E3 ligases with a range of selected transmembrane receptors, thereby inducing removal of these receptors from the cell surface. These findings also emphasize that not all combinations are effective.

Example 4

Materials and methods

Cell culture and transfection

Human Embryonic Kidney (HEK) 293T cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (GE Healthcare), 2mM UltraGlutamine (Lonza), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen). Cells were incubated at 37℃with 5% CO ₂ Is cultured in medium. Transfection was performed using FuGENE 6 (Promega) or Effectene (Qiagen) according to the manufacturer's protocol.

Constructs and antibodies

E6-Flag-Snorkel CKLF-like MARVEL transmembrane domain containing family protein 6 (CMTM 6) was obtained by subcloning using Q5High-Fidelity 2X Master Mix (NEB). Sequence verification was performed on the constructs. A mouse anti-Flag (Sigma-Aldrich) primary antibody was used for Immunofluorescence (IF). These primary antibodies were diluted according to the manufacturer's instructions. The secondary antibody for IF was used at 1:300 (Life technologies).

Immunofluorescence and confocal microscopy.

HEK293T cells were grown on laminin (Sigma-Aldrich) coated glass coverslips in 24-well plates. Six hours after transfection, cells were incubated overnight with 50nM bi-VHHs (VHH Alpha- (GGGGS) 3-VHH E6). After treatment, the cells were washed twice with warm medium and fixed in a solution containing 4% formaldehyde in 0.05M phosphate buffer (pH 7.4). Cells were blocked in PBS buffer containing 2% bsa for 30 min at Room Temperature (RT). Subsequently, cells were incubated with primary antibodies against Flag for 1 hour at RT, followed by incubation with secondary antibodies in blocking buffer for 1 hour at RT. Cells were mounted in a Prolong Diamond (Life technologies) and images were acquired using a LSM700 confocal microscope using a 5-fold objective. The images were analyzed and processed with ImageJ.

Results and discussion

RNF43 and RNF128 induced removal of multi-transmembrane receptor CMTM6 from the cell surface following forced dimerization with bispecific VHH.

To extend the applicability of the heterobifunctional molecules of the invention to type 2 or type 3 transmembrane proteins, we used Snorkel tags. The N-terminus or N-and C-termini of type 2 and type 3 transmembrane proteins, respectively, are located in the cell. To detect its surface expression, a Snorkel tag may be added to the intracellular N-terminal or C-terminal region, allowing the protein to be labeled and detected extracellularly without interfering with its structure or subcellular localization. The Snorkel tag consists of a transmembrane domain (TMD) flanked by a linker region and two epitope tags. Depending on the target multi-spanning protein, we incorporate either the Alpha-Myc tag of the multi-spanning E3 ligase or the E6-flag tag of the multi-spanning target protein (FIG. 7A). For example, in addition to the previously generated construct, we also generated E6-Flag-Snorkel-CMTM6. When co-expressed in HEK293T cells, CMTM6 localizes to the cell surface. After overnight treatment with bi-VHH, RNF43 and small amounts of RNF128 were able to remove CMTM6 from the surface when treated with biVHH (fig. 7B).

These findings expand the scope of use of heterobifunctional molecules (e.g., bi-VHHs) to deliberately dimerize various transmembrane E3 ligases with transmembrane receptors (e.g., CMTM 6) to induce removal of these receptors from the cell surface. Furthermore, these findings again underscores the need to screen for effective E3 ligase-target protein combinations, as not all combinations are effective.

Example 5

To verify the promising combinations presented in the above screen in a physiological environment, we will use CRISPR/Cas9 technology to generate (cancer) cell lines expressing endogenous tagged E3 ligase and target protein. We will use guide RNA in combination with donor DNA for Alpha-Myc or E6 Flag tags, which will help to insert these tags between the Signal Peptide (SP) and the coding sequence of the first mature amino acid in the endogenous site of the E3 ligase or target protein (fig. 7A). Using these cell lines, we will evaluate the removal of endogenous target proteins from the cell surface upon forced dimerization with bi-VHH by microscopy, FACS or western immunoblotting (fig. 7B).

Claims

1. A method for identifying an effective combination of a transmembrane E3 ubiquitin ligase and a membrane bound protein, wherein the combination is effective when the transmembrane E3 ubiquitin ligase is capable of reducing the surface level of the membrane bound protein, preferably by ubiquitination of the membrane bound protein, when bound simultaneously to a heterobifunctional molecule, and wherein the method comprises the steps of:

a) Providing a cell, wherein the cell expresses the transmembrane E3 ubiquitin ligase and the membrane-bound protein on its cell surface;

b) Exposing the cell to the heterobifunctional molecule, wherein the heterobifunctional molecule comprises:

i) A first binding domain capable of specifically binding to an extracellular portion of the transmembrane E3 ubiquitin ligase; and

ii) a second binding domain capable of specifically binding to an extracellular portion of the membrane-bound protein; and

c) Determining the surface level of said membrane-bound protein of said cell,

2. The method according to claim 1, wherein the membrane-bound protein is a transmembrane protein.

3. The method according to claim 1 or 2, wherein the transmembrane E3 ubiquitin ligase is selected from the group consisting of RNF43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF130 and RNF128.

4. The method according to any of the preceding claims, wherein at least one of the following:

-the transmembrane E3 ubiquitin ligase comprises a first extracellular non-native epitope tag, and wherein the first binding domain of the heterobifunctional molecule binds to the first non-native epitope tag; and

-the membrane-bound protein comprises a second extracellular non-native epitope tag, and wherein a second binding domain of the heterobifunctional molecule binds to the second non-native epitope tag.

5. The method according to claim 4, wherein said first non-natural epitope tag and said second non-natural epitope tag are different tags.

6. The method according to claim 4 or 5, wherein said first non-natural epitope tag is at least one of an alpha tag and an E6 tag, and/or wherein said second non-natural epitope tag is at least one of an alpha tag and an E6 tag.

7. The method according to any one of claims 4 to 6, wherein at least one of said first and said second non-native epitope tag is located at each of said transmembrane E3 ubiquitin ligase and said membrane-bound protein

i) An N-terminal;

ii) C-terminal; and/or

iii) The extracellular loop region of the cell is provided,

at least one of (a) and (b).

8. A method according to any one of the preceding claims, wherein the heterobifunctional molecule is a bispecific antibody, preferably a bispecific nanobody.

9. The method according to claim 8, wherein the first binding domain of the heterobifunctional molecule is an anti-Alpha VHH and the second binding domain is an anti-E6 VHH, or wherein the first binding domain of the heterobifunctional molecule is an anti-E6 VHH and the second binding domain is an anti-Alpha VHH.

10. The method according to any of the preceding claims, wherein the membrane bound protein comprises a third non-natural epitope tag, and/or wherein the transmembrane ubiquitin E3 ligase comprises a fourth non-natural epitope tag, preferably wherein the third epitope tag and/or the fourth epitope tag is at least one of a His tag, a FLAG tag and a myc tag.

11. A method according to any one of the preceding claims, wherein the cell surface level of the membrane bound protein in step c) is determined by detecting the protein on the cell surface, preferably by immunofluorescence.

12. The method according to any one of the preceding claims, wherein the combination is effective when the cell surface level of the membrane bound protein is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least about 95% compared to the cell surface level of the membrane bound protein prior to step b), preferably by at least about 60%, 70%, 80%, 90% or at least about 95% compared to the cell surface level of the membrane bound protein prior to step b).

13. The method according to any one of claims 4 to 11, wherein in step a) a first cell and a second cell are provided, wherein,

Said second cell expressing a second transmembrane E3 ubiquitin ligase and said first membrane bound protein on its cell surface,

wherein in step b) the first cell and the second cell are exposed to the heterobifunctional molecule, wherein the heterobifunctional molecule comprises:

i) A first binding domain capable of specifically binding to the first extracellular non-native epitope tag; and

ii) a second binding domain capable of specifically binding to an extracellular portion of the membrane-bound protein, preferably to the second non-native epitope tag; and is also provided with

Wherein the surface level of the membrane bound protein of the first cell and the second cell is determined in step c), and wherein the combination is effective when the cell surface level of the membrane bound protein in the first cell is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least about 95% compared to the cell surface level of the membrane bound protein in the second cell after step b).

14. The method according to claim 13, wherein a third, fourth or more cells are provided, expressing a third, fourth or more transmembrane E3 ubiquitin ligase and the first membrane bound protein, respectively, on their cell surfaces,

wherein the transmembrane E3 ubiquitin ligase is a different ligase comprising the same first extracellular non-native epitope tag,

and wherein the method is preferably performed in a multiplexed manner.

15. The method according to any of the preceding claims, wherein the decrease in surface level of the membrane-bound protein is determined by a decrease in the total amount of membrane-bound protein in the cell, preferably by microscopy, biochemical analysis and/or FACS.

16. The method according to any of the preceding claims, wherein the cell provided in step a) overexpresses, optionally permanently overexpresses, at least one of the transmembrane E3 ubiquitin ligase and the membrane-bound protein.

17. The method according to any of the preceding claims, wherein the cell provided in step a) expresses the transmembrane E3 ubiquitin ligase and the membrane-bound protein at endogenous levels.

18. The method according to claim 17, wherein in the cell provided in step a), the genomic sequence encoding the transmembrane E3 ubiquitin ligase has been modified to incorporate a sequence encoding the first non-natural epitope tag and optionally a fourth non-natural epitope tag.

19. The method according to claim 17 or 18, wherein in the cell provided in step a) the genomic sequence encoding the membrane bound protein has been modified to incorporate a sequence encoding the second and optionally a third non-natural epitope tag.

20. The method according to any of the preceding claims, wherein the heterobifunctional molecule comprises a peptide linker located between the first binding domain and the second binding domain, and wherein preferably the peptide linker is (GGGGS) n, wherein n is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably wherein n is 3 or 5.

21. A heterobifunctional molecule comprising a first binding domain and a second binding domain, wherein,

ii) the second binding domain is capable of specifically binding to a transmembrane protein,

and wherein the transmembrane E3 ubiquitin ligase and the transmembrane protein are an effective combination as determined in the method of any one of claims 1 to 20.

22. The heterobifunctional molecule of claim 21, wherein said molecule binds to an extracellular portion of said transmembrane E3 ubiquitin ligase and an extracellular portion of said transmembrane protein.

23. Heterobifunctional molecule according to claim 21 or 22, wherein the transmembrane protein is a receptor, preferably a receptor involved in at least one of cancer, autoimmune disease, neurological disorder and inflammatory disorder.

24. The heterobifunctional molecule of any one of claims 21 to 23, wherein the heterobifunctional molecule is a bispecific antibody, preferably a bispecific nanobody.

25. A heterobifunctional molecule according to any one of claims 21 to 24 for use as a medicament.